Proteins capable of regulating NF-κB, JNK and apoptosis pathways and methods of using the same

ABSTRACT

Testing methods are provided for determining whether given candidate compounds are effective for regulating NF-κB, JNK and apoptosis cell activities. The methods involve forming a mixture including a compound such as a proteinaceous specie containing two death effector domains (DEDs) or structural or functional homologs and analogs thereof, the candidate compound and a binding target capable of specifically binding to at least one of the DEDs. This mixture is incubated under conditions such that, but for the presence of the candidate compound, the cell activity takes place to a determinable extent. After incubation, the activity is determined and is compared with the determinable extent thereof in the absence of the candidate compound. The assays may be carried out intracellularly or in a cell-free assay. Methods for altering NF-κB, JNK and apoptosis activities in the cell are also provided, and comprise introducing into the cell an activity-regulating amount of a dual DEDs-containing proteinaceous specie.

RELATED APPLICATION

This is a division of application Ser. No. 09/074,044 filed May 7, 1998.

SEQUENCE LISTING

A printed Sequence Listing accompanies this application, and has alsobeen submitted with identical contents in the form of acomputer-readable ASCII file on a floppy diskette.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly concerned with test methods or assaysdesigned to determine whether a candidate compound has a significantregulating affect upon cell activities, namely NF-κB, JNK and apoptosis,so as to facilitate the discovery and/or design of therapeutic agents.The invention is also concerned with novel proteinaceous species usefulin such assays, and to methods for regulating cellular NF-κB, JNK andapoptosis activities for therapeutic purposes.

2. Description of the Prior Art

Nuclear factor-kappa B (NF-κB) is a critical transcription factor whichis required for regulated expression of several genes involved ininflammation and immune response. Five known members of this family havebeen characterized to date and include c-Rel, p52, RelA and RelB. NF-κBis present in the cytoplasm of various cells as an inactive complex withan inhibitory protein called IκB. Stimulation by a number of agentsresults in the degradation of IκB and subsequent release of NF-κB. Oncereleased, NF-κB is free to migrate to the nucleus and bind to thepromoter of specific genes possessing its cognate binding site.

A large number of endogenous and exogenous conditions can lead to theactivation of the NF-κB pathway. These include viral and bacterialinfections, environmental toxins, UV irradiation, inflammatory cytokines(TNFα, TNFβ, IL- 1, IL-17, LIF, etc.), growth factors (IL-2, insulin,M-CSF, PDGF, NGF, etc.), immunoreceptor ligands (e.g., CD3 ligand, CD2ligand, CD 28 ligand, CD30 ligand, CD 40 ligand, etc.), inflammatorymediators (e.g., Thrombin, angiotensin II, Leukotriene B4), celladhesion molecules, and several stressful situations (e.g., hypoxia,osmotic shock, hemorrhage, etc.)

Activation of NF-κB leads to the transcriptional activation of severalgenes which play a crucial role in both innate and acquired immuneresponses. Some of the noteworthy genes activated by NF-κB include thosefor cytokines and growth factor such as TNFα, TNFβ, IL-1β, IL-2, I,IL-3, IL-6, IL-8, IL-12, GM-CSF, G-CSF, chemokines, cell adhesionmolecules, acute phase proteins and transcription factors p53 and c-myc.In addition, several viruses are activated by NF-κB including the Humanimmunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV).

Given the large number of inducers of NF-κB and equally large number ofits target genes, it is not surprising that activation of NF-κB has beenimplicated in the pathogenesis of many acute and chronic inflammatoryconditions, such as septic shock, rheumatoid arthritis, Crohn's Diseaseand atherosclerosis. In addition, NF-κB has a role in oncogenesis andviral transcription regulation, such as in HIV, adenoviruses and papovaviruses.

A large number of molecules with immunosuppressive and anti-inflammatoryproperties have been studied as inhibitors of NF-κB. These includeglucocorticoids and other steroid hormones, cyclosporin A, FK506,rapamycin, salicylates and gold compounds. All these compounds arecurrently being used for the treatment of several human autoimmune andinflammatory disorders which underscores the clinical and commercialimportance of NF-κB pathway in the control of inflammation and immuneresponse. However, there are two major problems with the aboveinhibitors. First, the majority of these compounds have broad range ofactivities so that they will suppress NF-κB activation by a large numberof stimuli. This manifests itself in the form of severeimmunosuppression and resulting pre-disposition to opportunisticinfections. Second, the majority of these agents have numerous adverseeffects associated with the use of these compounds.

Mammalian cells respond to extra cellular stimuli by activation of theMAP kinase family of signaling proteins. Members of the JNK subgroup ofMAP kinases are activated in response to diverse extracellular stimuli,including UV irradiation, proinflammatory cytokines and certainmitogens. The JNKs in turn phosphorylate and activate the transcriptionfactor c-Jun, an important component of the transcription activatorAP-1. Activation of the JNK pathways has been implicated in a widevariety of responses ranging from cell growth, proliferation,differentiation, cell death, andprotection from cell death. As such,this pathway has been implicated in the pathogenesis of humanmalignancies as well as diseases associated with abnormal cell death.The JNK pathway is activated by several members of the TNFR family. Theroll of this pathway and the mediation of cell death by the TNFR familymembers is still a subject of controversy.

Apoptosis or programmed cell death plays an essential role in the normalgrowth and development. Abnormalities in this pathway have been linkedto the pathogenesis of a number of diseases. For example, failure ofcells to undergo apoptosis has been associated with the development of alarge number of malignancies, especially low grade lymphomas. Similarly,defects in the apoptosis induced by members of the TNFR family ofproteins have been linked to several autoimmune diseases. On the otherhand, a large number of diseases are characterized by excessiveapoptosis including Alzheimer's disease, AIDS, osteoporosis, ischemicinjury, myocardial infarction, and hepatic necrosis. Owing to thecentral role played by apoptosis in the pathogenesis of several humandiseases, the apoptosis pathway and therapies that can modulate thispathway are the focus of extensive research.

Some of the proteins involved in apoptosis have been identified and someinteractions among these proteins have been described. However, themechanisms by which these proteins mediate their activity remainsunknown. Given the importance of cell apoptosis and the potentialbenefits which would flow from effective regulation thereof,considerable research has been undertaken to elucidate the involvedproteins and pathway.

Kaposi Sarcoma (KS) is the most common malignancy found in patients withHIV infection. Recent studies have implicated a herpes virus termedKaposi Sarcoma associated Herpes Virus (KSHV/HHV8) in the pathogenesisof this disease as well as several other malignancies. The mechanism bywhich KSHV causes malignant transformation is unclear.

Molluscum Contagiosum Virus (MCV) is a benign skin tumor caused by apoxvirus. A hallmark of infection by this virus is an almost completelack of inflammation in the affected areas which allows the infection topersist for months or even years and to recur after treatment. Themechanism by which MCV blocks the local immune and inflammatory responseis unknown.

SUMMARY OF THE INVENTION

The present invention is predicated upon the discovery that certainknown molecules as well as novel mutants thereof modulate the signaltransduction associated with NF-κB, JNK and apoptosis pathways. In lightof this discovery, a number of important screening techniques have beendeveloped, and therapies, including the development of usefulpharmacological agents, can be foreseen.

In particular, it has been found that known protein molecules such asCaspase 8, Caspase 10, MRIT-α1, MC159L, MC160L, E8, the N-terminalprodomains of Caspase 8, Caspase 10 and MRIT-α1 and structural orfunctional homologs and analogs of the foregoing can regulate (i.e.,either enhance or inhibit) the NF-κB, JNK and apoptosis pathways. Theseproteinaceous species are preferably characterized by the presence of anN-terminal prodomain comprising two death effector domains (DEDs) whichdirectly interact with the TRAF family of adaptor proteins andserine-threonine family of protein kinases in order to regulate thepathways. For example, Caspases 8 and 10 activate the NF-κB and JNKpathways; similarly, MRIT-α1, a proteolytically inactive Caspase 8homolog and K13-ORF, activate both the NF-κB and JNK pathways. Theinteractions of Caspase 10 with TRAF2 and TRAF5 have been found topro-apoptotic.

In one aspect of the invention, a method is provided for the testing ofcandidate compounds such as pharmacological agents and lead compoundstherefor. Broadly speaking, such a method involves forming in a cell amixture including a proteinaceous specie containing two death effectordomains (DEDs), the candidate compound, and a binding target proteincapable of specifically binding with at least one of the DEDs. Thismixture is incubated under conditions such that, but for the presence ofthe candidate compound, a cell activity selected from the groupconsisting of NF-κB, JNK and apoptosis takes place to a determinableextent. Finally, the activity is detected and is compared with thedeterminable extent of the activity in the absence of the candidatecompound. This comparison affords a measure of the effectiveness of thecandidate compound in regulating the cell activity in question.

Preferably, the proteinaceous specie is formed in situ within the cellby inserting into the cell an expression vector for the proteinaceousspecies, and causing the vector to express the specie within the cell.Moreover, a reporter gene construct is preferably inserted into the cellhaving a reporter gene operably linked with a promoter and responsive tothe activity; the reporter gene expresses a detectable protein inresponse to the activity, and the extent of expression of the detectableprotein is measured as a measurement of the cell activity. For example,a luciferase reporter construct may be used to good effect in the assaysof the invention, and detection methods for luciferase.

The preferred proteinaceous species in accordance with this aspect ofthe invention comprises a protein fragment having at least about 40amino acids (more preferably about 50 amino acids) and wherein each ofthe DEDs therein respectively has at least about 20% homology (i.e.,amino acid residue identity) to any of the DED1 or DED2 domains of SEQID Nos. 1-16, inclusive. More preferably, the degree of homology is atleast about 50%, and more preferably at least about 90%. Comparison forhomology among protein fragments is usually performed with sequencesbetween about 6 and 500 residues, preferably between about 10 and 100residues and more preferably between about 25 and 35 residues.Comparisons for substantial similarity can be performed using anytechniques known in the art. Furthermore, limited modifications can bemade to the sequences without destroying the biological function of theDEDs-containing proteinaceous species because only a portion of theentire primary sequence is required in order to affect activity. Forexample, genetically engineered fragments of DEDs-containing proteins,either alone or fused to heterologous proteins, that retain certainactivities (i.e., measurable NF-κB, JNK, or apoptosis modulatingactivity; specific binding ability to TRAFs, serine-threonine proteinkinases, etc.) fall within the definition of the DEDs containingproteinaceous species claimed as such. An analysis of the conservedamino profiles of the DEDs confirms that each of the DEDs advantageouslycomprises the sequence X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ LL, where X₁ is Eor H, X₂ is L, C, M or I, X₃ is L or F, X₄ is Y, F, C, A or E, X₅ is R,V, A, I or S, X₆ is S, L, I, or V, X₇ is R, N, Q or G, X₈ is R or Q, X₉is F, R, L, M, K, H or P, and X₁₀ is D or K. The proteinaceous speciesshould moreover have a molecular weight of from about 5-200 kDa.

In other aspects of the invention, a similar assay method is providedwhich involves forming a mixture (which may be cell free) including aproteinaceous specie containing two DEDs and selected from the groupconsisting of K13-ORF, E8, MC159L, MC160L, Caspase 8, Caspase 10, theN-terminal prodomains of Caspase 8, Caspase 10 and MRIT-α1 andstructural or functional homologs and analogs of the foregoing. Themixture further has the candidate compound and a binding target capableof binding with at least one of the DEDs and selected from the groupconsisting of the TRAF proteins. This mixture is incubated underconditions such that, but for the presence of the candidate compound,the binding target-DED specific binding takes place to a determinableextent. Thereafter, the binding target-DED specific binding is detectedand is compared with the determinable extent of the binding in theabsence of the candidate compound. A similar assay is provided whereinthe binding target protein is selected from the group consisting of theserine-threonine kinase proteins, and in such case the proteinaceousspecie may be any such specie containing two DEDs.

A variety of cell-free assay techniques may be used in this aspect ofthe invention, e.g., electrophoretic mobility shift assays, in vitroimmunoassays and protein-protein binding assays. Alternately, the assaymay be carried out intracellularly, in a manner identical to thatdescribed previously; likewise, the assays may be qualitative orquantitative depending upon the desires of the user. Finally, theattributes of the DEDs in the proteinaceous species of this aspect ofthe invention are identical to those set forth above.

It is to be understood that intracellular assays in accordance with theinvention may be carried out without actual addition of the mixturecomponents, i.e., the binding target protein may be (and normally is) anaturally occurring cell protein. Thus, the step of forming a mixture ina cell may involve inserting into the cell an expression vector for theproteinaceous specie, actual addition of the candidate compound, and useof a naturally occurring binding target protein within the cell.Moreover, while mammalian cells are often preferred in the assays of theinvention, other types of cells, for example yeast cells may be used.

The assays of the present invention provide efficient methods foridentifying pharmacological agents or lead compounds therefor, and asindicted generally involved assaying for compounds which in some mannerregulate or modulate a cell activity. The methods are amenable tocost-effective high throughput drug screening and have immediateapplicability in a broad range of domestic and internationalpharmaceutical and biotechnology drug development programs. Asunderstood by those skilled in the art, normally a plurality of assaymixtures are run in parallel with different candidate compoundconcentrations to obtain a differential response at variousconcentrations. Typically, a control free of the candidate compound butotherwise identical is run simultaneously with the candidatecompound-containing assay mixtures.

The assays of the invention may be quantitative, i.e., a quantitativemeasurement of cell activity is taken after the incubation step, andthis is compared with a quantitative determination of activity in theabsence of the candidate compound. Such an assay therefore gives aquantitative measure of the regulation effectiveness of the candidatecompound. Alternately, the assays hereof may be qualitative, involvingonly a qualitative ascertainment of the extent of activity afterincubation, in comparison to a qualitative determination of the extentof activity in the absence of the candidate compound.

Candidate compounds encompass numerous chemical classes such as organiccompounds having a molecular weight of from about 50-2500 (preferablyless than about 1000), proteinaceous species such as proteins, peptidesand polypeptides, saccharides, fatty acids, sterols, isoprenoids,purines, pyrimidines, as well as derivatives and structural orfunctional analogs of the foregoing. Candidate compounds may be obtainedfrom a wide variety of sources including libraries of synthetic ornatural compounds. For example, numerous means are available for randomand directed synthesis of a wide variety of organic compounds andbiomolecules, including expression of randomized oligonucleotides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural and synthetically produced libraries and compoundsare readily modified through conventional chemical, physical, andbiochemical means. In addition, known pharmacological agents may besubject to directed or random chemical modifications, such as acylation,alkylation, esterification, amidification, etc., to produce structuralanalogs.

A variety of other reagents may also be included in the assay mixtures,such as salts, buffers, neutral proteins, detergents and the like. Suchagents may be used to facilitate desired binding or to reducenon-specific or background interactions. Also, reagents that otherwiseimprove the efficiencies of the assays may be used, such as proteaseinhibitors and antimicrobial agents.

Incubation of the assay mixtures is carried out under conditionswhereby, but for the presence of the candidate compound, the cellactivity and/or binding target-DED specific binding takes place to areadily determinable extent. The mixture components can be added in anyorder in the in vitro assays, so long as the requisite binding isallowed to occur. Generally speaking, incubations are carried out at atemperature of from about 4-40° C., more commonly between 15-40° C.Incubation periods normally range between 0.1-36 hours and arepreferably less than 5 hours.

After incubation, the extent of measurable activity can be detected byany convenient technique. For cell-free binding type assays, aseparation step is often used to separate bound from unbound components.Those skilled in the art are familiar with a number of ways suchseparations may be effected. Actual detection can be accomplished bydirectly or indirectly measuring a detectable byproduct of the assay,such as a detectable enzyme in the case of cellular assays. Forcell-free binding assays, one of the components usually comprises or iscoupled to a detectable label, be it radioactive, luminescent oroptical. Again, skilled artisans are familiar with numerous techniquesfor detecting such labels.

The invention also provides new effective mutants having the sequencesof SEQ ID Nos. 26-28. These mutants may be isolated or generatedintracellularly as described.

The invention further includes a method for regulating a cell activityselected from the group consisting of NF-κB, JNK and apoptosiscomprising the step of introducing into a living cell anactivity-regulating amount of a proteinaceous specie not otherwisepresent in the living cell, the specie taken from the group consistingof Caspase 8, Caspase 10, MRIT-α1, K13-ORF, MC159L, MC160L, E8, theN-terminal prodomains of Caspase 8, Caspase 10 and MRIT-α1 andstructural or functional homologs and analogs of the foregoing.

Such therapeutic applications are normally local, involvingadministration of a selected agent at a site of interest. Varioustechniques can be used for providing the therapeutic agent at a selectedsite, such as injection, use of catheters, trocars, projectiles,pluronic gel, stents, sustained drug release polymers, or otherexpedient which provides for internal access.

As used herein, the term "structural or functional homologs and analogsand analogs" in reference to a proteinaceous specie or binding target isintended to mean species having similar, non-identical sequence(s)considered by those skilled in the art to be functionally equivalent tothe specific proteinaceous specie or binding target in question, as wellas non-proteinaceous compounds such as synthetic analogs or mimics whichhave the same functional properties in the methods of the invention asthe DEDs-containing proteins or binding targets, as the case may be. Forexample, the sequence of a given proteinaceous specie or binding targetmay be altered by substitution, deletion or addition of amino acidresidues which have no, or very little, effect upon the functionality ofthe specie or target. Similarly, spliced isoforms and active fragmentshaving the desired functionality may be used. Functionally equivalentsynthetic mimics are known in the art and could be used in lieu ofactual proteinaceous species or binding targets. All such alterationsand substitutions are intended to be encompassed as "structural orfunctional homologs and analogs." The term "isolated" in reference to aproteinaceous specie means a specie that is relatively free fromcontaminating lipids, polypeptides, nucleic acids or other cellularmaterial which may be associated with the specie in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the extent of expression of luciferasereporter enzyme in a series of intracellular tests wherein respectiveCaspase 8 constructs were expressed in 293T cells to determine theireffect upon NF-κB pathway activity, as compared with a null vectorcontrol;

FIG. 2 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in a series of intracellular tests wherein Caspase 8 and aCaspase 8 mutant (C360S) were expressed in MCF-7 human breast cancercells to determine their effect upon NF-.sub.κ B pathway activity, ascompared with a null vector control;

FIG. 3 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in a series of intracellular tests wherein Caspase 8,Caspase 10 Mch4 isoform and a Caspase 10 mutant (C358A) were expressedin 293T cells to determine their effect upon NF-κB pathway activity, ascompared with a null vector control;

FIG. 4 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in an intracellular test wherein Caspase 10 FLICE2 wasexpressed in 293T cells to determine their effect upon NF-κB pathwayactivity, as compared with a null vector control;

FIG. 5 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in a series of intracellular tests wherein variousCaspases were expressed in 293T cells to determine their effect uponNF-κB pathway activity, as compared with a null vector control;

FIG. 6 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in a series of intracellular tests wherein MRIT-α1 andMRIT-β1 isoforms of MRIT were expressed in 293T cells to determine theireffect upon NF-κB pathway activity, as compared with a null vectorcontrol;

FIG. 7 is a photograph illustrating electrophoretic gel mobility shiftassay test results wherein the effect of expression of Caspase 8 mutant(C360S), Caspase 10 mutant (C358A), MRIT-α1, FAS/CD95, and TNFR1 wasdetermined as compared with a null vector control;

FIG. 8 is a graph similar to that of FIG. 1 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofcrmA, p35 and DN-mTRAF2 on NF-κB activity induced by Caspase 8 mutant(C360S), Caspase 10 mutant (C358A) and MRIT-α1 in 293T cells wasmeasured, as compared with a null vector control;

FIG. 9 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofDN-mTRAF2 and DN-mTRAF5 on NF-κB activity induced by Caspase 8, MRIT-α1and DR3 in 293T cells was measured, as compared with a null vectorcontrol;

FIG. 10 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofmTRAF1 and I-TRAF on NF-κB activity induced by Caspase 8, MRIT-α1 andDR3 in 293T cells was measured, as compared with a null vector control;

FIG. 11 is a homology matrix depicting the degrees of homology betweenthe DED1 and DED2 domains of MRIT-α1, K13-ORF, E8, MC159L, MC160L,Caspase 8, Caspase 10, FADD and PEA-15;

FIG. 12 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofND-Caspase 8, Caspase 8 D73A and Caspase 8 L74A on NF-κB activityinduced by Caspase 8 mutant (C360S), MRIT-α1 and DR3 in 293 T cells wasmeasured, as compared with a null vector control;

FIG. 13 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofCaspase 8 D73A, Caspase 8 L74A and Caspase L75A on NF-κB activitymediated by TNFR1, Fas, DR3, DR4 and DR5 in 293T cells was measured, ascompared with a null vector control;

FIG. 14 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in an intracellular test wherein the effect of DN-mTRAF2,I-TRAF, crmA, p35 and DN-mTRAF5 on NF-κB activity mediated by K13-ORF in293T cells was measured, as compared with a null vector control;

FIG. 15 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in an intracellular test wherein the effect of MC159L onNF-κB activity mediated by Caspase 8 mutant (C360S), Caspase 10 mutant(C358A) and MRIT-α1 in 293T cells was measured, as compared with a nullvector control;

FIG. 16 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in an intracellular test wherein the effect of E8 on NF-κBactivity mediated by Caspase 8 mutant (C360S), Caspase 10 mutant (C358A)and MRIT-α1 in 293T cells was measured, as compared with a null vectorcontrol;

FIG. 17 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofCaspase 8 mutant (D73A), MC159L and PEA-15 on NF-κB activity induced byTNFR1, Fas/CD95, DR3, DR4, TNFR2, CD40, CD30 and LTBR in 293T cells wasmeasured, as compared with a null vector control;

FIG. 18 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofCaspase 8 mutant (D73A), MC159L and PEA-15 on NF-κB activity induced byK13-ORF in 293T cells was measured, as compared with a null vectorcontrol;

FIG. 19 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofCaspase 8 mutant (D73A), Caspase 8 mutant (L75A), MC159L and PEA-15 onNF-κB activity induced by RIP in 293T cells was measured, as comparedwith a null vector control;

FIG. 20 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofCaspase 3 on NF-κB activity induced by Caspase 8 mutant (C360S), MRIT-α1and DR3 in 293T cells was measured, as compared with a null vectorcontrol;

FIG. 21 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofmTRAF1, A20, Caspase 7 mutant (C1865) and Caspase 9 mutant (C288S) onNF-κB activity induced by Caspase 8 mutant (C360S), Caspase 10 mutant(C358A), MRIT-α1 and K13-ORF in 293T cells was measured, as comparedwith a null vector control;

FIG. 22 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofnon DEDs-containing peptides Caspase 7 mutant (C186S) and Caspase 9mutant (C288S) on NF-κB activity induced by TNFR1, Fas/CD95, DR3 and DR4in 293T cells was measured, as compared with a null vector control;

FIG. 23 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofDN-NIK on NF-κB activity induced by Caspase 8 mutant (C360S), Caspase 10mutant (C358A) and MRIT-α1 in 293T cells was measured, as compared witha null vector control;

FIG. 24 is a graph illustrating the expression of luciferase in a seriesof intracellular tests wherein the effect of Caspase 8 mutant (C360S),Caspase 10 mutant (C358A), MRIT-α1, Caspase 8 prodomain, Caspase 10prodomain, MRIT-β1 and CD40 on JNK pathway activation in 293EBNA cellswas measured, as compared with a null vector control;

FIG. 25 is a photograph illustrating a JNK activation assay based uponc-jun phosphorylation wherein the effect of expression of Caspase 8mutant (C3 60S), Caspase 10 mutant (C358A), MRIT-A 1 and CD40 wasdetermined as compared with a null vector control;

FIG. 26 is a graph similar to that of FIG. 8 illustrating the expressionof luciferase in a series of intracellular tests wherein the effect ofDN-mTRAF2, I-TRAF, Caspase 8 mutant (D73A) and Caspase 8 mutant (L75A)on JNK activity induced by Caspase 8 mutant (C360S), Caspase 10 mutant(C358A) and MRIT-α1 in 293EBNA cells was measured, as compared with anull vector control;

FIG. 27 is a graph similar to that of FIG. 26 illustrating theexpression of luciferase in a series of intracellular tests wherein theeffect of Caspase 8 mutant (D73A) on JNK activity induced by TNFR1,Fas/CD95, DR3, DR4 and CD40 in 293T cells was measured, as compared witha null vector control;

FIG. 28 is a pair of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 8 and mTRAF 1-HA, and between Caspase 8 and mTRAF2-FLAG, weremeasured in 293T cells as compared with a control;

FIG. 29 is a pair of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interactions betweenCaspase 8 mutant (C360S) and TRAF3-HA/GFP-HA, and between Caspase 7mutant (C186S) and TRAF3-HA/GFP-HA, were measured in 293T cells ascompared with a control, wherein the lysate lane shows the expression ofthe HA-tagged proteins in the total cellular extract;

FIG. 30 is a pair of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 8 and TRAF5 FLAG, and between Caspase 8 and DN-TRAF5 FLAG, weremeasured in 293T cells as compared with a control;

FIG. 31 is a series of three photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 8 protease domain FLAG (a.a. 217-479) and mTRAF1-HA, betweenCaspase 8 prodomain FLAG (a.a. 1 -180), and between MRIT-β1 FLAG (a.a.1-221), was measured in 293T cells as compared with a control, whereinthe lysate lane shows the expression of the HA-tagged proteins in thetotal cellular extract;

FIG. 32 is a pair of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 8 protease FLAG (a.a. 217-479) and mTRAF2-HA/GFP-HA, and betweenCaspase 8 prodomain FLAG (a.a. 1- 180) and mTRAF2-HA/GFP-HA, wasmeasured in 293T cells as compared with a control;

FIG. 33 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 8 FLAG (a.a. 1-180) and mTRAF1-HA, mTRAF2-HA and FADD-AU1, wasmeasured in 293T cells as compared with a control;

FIG. 34 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenCaspase 7 mutant (C186S) FLAG and NIK-HA and mTRAF1-HA/GFP-HA wasmeasured in 293T cells as compared with a control, wherein the lysatelane shows the expression of the HA-tagged proteins in the totalcellular extract;

FIG. 35 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenmTRAF2-HA and K13-ORF FLAG, MC159L FLAG, MRIT-β1 FLAG and PEA-15 FLAGwas measured in 293T cells;

FIG. 36 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interaction betweenNIK-HA and Caspase 8 mutant (C360S) FLAG, Caspase 8 protease FLAG,Caspase 10 mutant (C358A) FLAG, MRIT-α1 FLAG, Caspase 7 mutant (C186S)FLAG, K13-ORF FLAG and PEA-15 FLAG was measured in 293T cells, ascompared with a control, wherein the lysate lane shows the expression ofthe HA-tagged proteins in the total cellular extract;

FIG. 37 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the effect of mTRAF1-HAon interactions between NIK-HA and Caspase 8 FLAG was measured in 293Tcells, as compared with a control and a lysate control;

FIG. 38 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the effect of mTRAF1-HAon interactions between NIK-HA and Caspase 8 mutant (C360S) FLAG wasmeasured in 293T cells;

FIG. 39 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interactions betweenRIP-HA and Caspase 8 mutant (C360S) FLAG, Caspase 10 mutant (C358A)FLAG, MRIT-α1 FLAG, Caspase 8 prodomain FLAG and MRIT-β1 FLAG wasmeasured in 293T cells, as compared with a control;

FIG. 40 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interactions betweenCaspase 8 mutant (C360S) and IKK1 FLAG and IKK2 FLAG was measured in293T cells, as compared with a control;

FIG. 41 is a series of photographs depicting the results of aco-expression-immunoprecipitation assay wherein the interactions betweenCaspase 8 and mTRAF 1-HA and mTRAF2-FLAG were measured in a cell-freesystem;

FIG. 42 is a graph illustrating the results of a series of apoptoticcell experiments wherein the effect of mTRAF2 and mTRAF2+crmA upon celldeath induced by Caspase 8, Caspase 8 prodomain (a.a. 1-180) and Caspase8 mutant (C360S) in 293T cells was measured;

FIG. 43 is a graph illustrating the results of a series of apoptoticcell experiments wherein the effect of I.sub.κ B-ΔN and JIP upon celldeath induced by Caspase 10 Mch4 isoform and Caspase 9 in 293T cells wasmeasured, as compared with a control; and

FIG. 44 is a graph illustrating the results of a series of apoptoticcell experiments wherein the effect of mTRAF1, mTRAF2, TRAF3, TRAF5 andTRAF6 upon cell death induced by Caspase 10, Caspase 7 and Caspase 9 in293T cells was measured, as compared with a control.

BRIEF DESCRIPTION OF THE SEQ IDS

SEQ ID Nos. 1-16, inclusive are respectively the DED1 and DED2 sequencesfor MRIT, K13-ORF, E8, MC159L, MC160L, Capase 8, Capase 10, FADD andPEA-15;

SEQ ID Nos. 17-25, inclusive, are respectively the entire prodomainsequences for MRIT, Caspase 8, FADD, K13-ORF, MC159L, MC160L, E8,PEA-15, and Caspase 10; and

SEQ ID Nos. 26-28, inclusive, are respectively the sequences for themutants for Caspase 8 (D73A), Caspase 8 (L74A) and Caspase 8 (L75A).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred procedures and proteinaceousspecies useful in the context of the invention. It is to be understood,however, that these examples are provided by way of illustration onlyand nothing therein should be taken as a limitation upon the overallscope of the invention.

Materials

293T and MCF7 cells were obtained from Dr. David Han (University ofWashington, Seattle, Wash.). 293EBNA cells were obtained fromInvitrogen. All cells were maintained in DMEM media with 10% fetal calfserum at 37 C with 5% carbon dioxide.

Rabbit polyclonal antibodies against FLAG, HA and myc-tags were obtainedfrom Santa Cruz Laboratories. Antibodies against the AU1 epitope-tagswere obtained from Babco. Myc beads and Flag beads were obtained fromSanta Cruz Laboratories, Santa Cruz, Calif. and Kodak Scientific ImagingSystems, New Haven, Conn., respectively.

Methods--Expression constructs

All cDNA clones refer to the human sequences unless specified otherwise.The sequences and/or expression constructs for cDNAs clones encoding thefollowing molecules have been previously described in the listedreferences: Caspase 8 (FLICE; GenBank U58143) (Muzio et al., Cell,85:817-827, 1996); Caspase 7 (ICE-LAP3; GenBank U39613) (Duan et al., J.Biol Chem, 271:1621-1625, 1996); Caspase 10 (Mch4; GenBank Q92851) andits active site mutant (i.e. Caspase 10 C358A) (Fernanes-Alnemri et al.,Proc. Nat'l Acad Sci U.S.A., 93:7464-7469, 1996); Caspase 10 (FLICE2)(Vincenz et al., J Biol Chem, 272:6578-6583,1997); Caspase 9 (ICE-LAP6;GenBank P55211) (Duan et al., J Biol Chem, 271:16720-16724, 1996);Caspase 3 (YAMA; GenBank P42574) (Tewari et al., Cell,. 81:801-809,1995); Caspase 2--GenBank P42575 (Wang et al., Cell, 78:739-750,1994);Caspase 1--GenBank P89116 (Thornberry et al., Nature,356:768-764,1992);FADD--GenBank Q13158 (Chinnaiyan et al., Cell, 81:505-512; 1995); E8(PIR Database S55668) and MC159L (GenBank U60315) (Hu et al., J BiolChem, 272:9621-96214, 1997); TNFR1 (GenBank P19438) and DR3 (GenBankU83597) (Chinnaiyan et al., Science, 274:990-992, 1996); crmA, cowpoxserpin, (Tewari et al., J Biol Chem, 270:3255-3260, 1995); CD40 (GenBankP25942) (Stamenkovic et al., Embo J, 8:1403-1410,1989); NIK(NF-κB-inducible kinase, GenBank Y10256), NIKΔ1234 and NIKΔ2101(dominant negative NIK) (Natoli et al., J Biol Chem, 272:26079-26082,1997); mTRAF1 (GenBank P39428) and mTRAF2 (GenBank P39429) (Rothe etal., Cell, 78:681-692,1994); TRAF3 (GenBank U15637) (Cheng et al.,Science, 267:1494-1498; 1995); mTRAF5 (GenBank D83528) (Nakano et al., JBiol Chem, 271:14661-14664,1996); A20 (Sarma et al., J Biol Chem,270:12343-12346, 1995); I-TRAF (Rothe et al., Proc Nat'l Acad SciU.S.A., 93:8241-8246, 1996); IKK1 (GenBank AF009225, IKK2 (GenBankAF029684) and their mutants (Nakano et al., Proc Nat'l Acad Sci U.S.A.,95:3537-3542, 1998); DN-IκBα or IκBα-ΔN (missing the N-terminal 36 aminoacids) (Brockman et al., Mol Cell Biol, 15:2809-18; 1995); NF-κB drivenluciferase reporter construct (Berberich et al., J Immunol, 153:4357-66;1994); and, JIP (Dickens et al., Science, 277:693-696,1997). Theseconstructs were either used directly or further modified to addappropriate epitope-tags using PCR and standard molecular biologycloning techniques as described in Molecular Cloning, 2d Ed. bySambrook, Fritsch, Maniatis, Cold Spring Harbor Laboratory Press, 1989,incorporated by reference herein. An RSV promoter driven β-galactosidasereporter construct was a gift of Dr. Mark Kay (University ofWashington).

Constructs encoding K13-ORF (GenBank U90534) were prepared by PCRamplification of the desired coding sequence from a human genomic DNAsample containing KSHV/HHV8 (human herpesvirus 8) genomic DNA and wasobtained from Dr. Tim Rose (University of Washington, Seattle, Wash.).Primers for PCR amplification were based on the published sequence ofK13-ORF and carried additional sequences at their 5' end for subsequentrestriction digestion and cloning of the amplified insert. Constructsencoding p35 were similarly prepared by using a bacuolovirus vectorpFastBac HTa (Life Technologies, Inc., catalog no. 10584-027) DNA as thetemplate. Constructs encoding mTRAF1, mTRAF2, TRAF3, I-TRAF, and PEA-15(GenBank Q15121), mTRAF5, and Lymphotoxin β Receptor (LBPR) weresimilarly prepared using IMAGE consortium EST clones as templates:

    ______________________________________                                               mTRAF1 636225                                                                         mTRAF2 439083                                                    mTRAF5 568002                                                                 TRAF3 290035                                                                  I-TRAF 638576                                                                 PEA-15 361256                                                                 LTBR 810443                                                                 ______________________________________                                    

All EST clones were obtained from the Genome Systems, Inc., St. Louis,Mo.

Myc-epitope tagged constructs encoding CD40 (a.a. 16 to 277), and LTBR(Gen Bank P36941) (a.a. 28-435) were similarly prepared by amplifyingthe corresponding inserts using custom primers and using a CD40 cDNA andIMAGE consortium EST clone as templates respectively. The amplifiedproducts were subsequently cloned in to a modified pSecTag A vector(Invitrogen) containing a DNA segment encoding a Myc-epitope(EQKLISEEDL) downstream and in-frame with a murine Ig κ-chain signalpeptide. Myc-tagged DR3, DR4 (GenBank U90875), DR5 (GenBank AF016268)and Fas were similarly constructed as previously described (Chaudhary etal., Immunity, 7:821-830; 1997). A construct encoding RIP-HA has alsobeen described previously (Chaudhary et al., Immunity, 7:821-830, 1997).The expression constructs encoding His-tagged MRIT-α1(GenBank U85059)and MRIT-β1 (GenBank Y14040) are described in Han et al., Proc Nat'lAcad Sci U.S.A., 94:11333-11338 (1997). Unless specified otherwise, theconstruct MRIT refers to the MRIT-α1 isoform. Constructs encodingFLAG-TRAF6 and FLAG-CD28/MTRAF5 were as described in Duckett et al.,Genes Dev 11:2810-21 (1997). Unless specified otherwise, the variousepitope-tagged mammalian expression constructs were constructed in thepcDNA3 expression vector (Invitrogen). All TRAF5 constructs refer to themurine TRAF5 cDNA clone. Although murine clones for TRAF5, TRAF1 andTRAF2 have been used in this invention, the corresponding human clonesare likely to behave in a similar fashion. Similarly, catalyticallyactive site mutant proteins for Caspases (i.e., Caspase 8 C360S, Caspase10 C358A, Caspase 9 C288S, and Caspase 7 C186S) were used for assaysinvolving activation of NF-κB and JNK pathways and for binding studies.This was done for the ease of experimental design based on the lack ofcytotoxicity of these proteins. It is expected that the correspondingwild-type Caspases will have similar properties in the above studies.

C-terminal FLAG Epitope Expression Vectors

The C-terminal FLAG epitope expression vectors encoding Caspase 8,Caspase 8 C360S, Caspase 8 prodomain, Caspase 8 protease domain, Caspase3, MRIT-α1, MRIT-β1, mTRAF2, mTRAF5, RIP (GenBank Q13546), K13-ORF,MC159L, E8 and PEA-15 were constructed by joining in-frame a DNA segmentencoding the following amino acid sequence to the 3' end of theprotein-coding sequences of cDNAs encoding the above proteins:ETDFYDYKDDDDK.

C-terminal HA Epitope Expression Vectors

The C-terminal HA (Heamagglutinin) epitope expression vectors encodingmTRAF1, mTRAF2, I-TRAF, TRAF3, mTRAF5, RIP, K13-ORF, MC159L, E8 andPEA-15 were constructed by joining in-frame a DNA segment encoding thefollowing amino acid sequence to the 3' end of the protein-codingsequences of the cDNAs encoding the above proteins: ETDFYPYDVPDYA

C-terminal myc Epitope Expression Vectors.

The C-terminal myc epitope expression vectors encoding Caspase 8,Caspase 8 C360S, Caspase 8 prodomain, and Caspase 8 protease domain wereconstructed by joining in-frame a DNA segment encoding the followingamino acid sequence to the 3' end of the protein-coding sequences of thecDNAs encoding the above proteins: ETDFYEQKLISEEDL.

Generation of Various Other Expression Vectors

To generate the expression vectors encoding the DED1 (amino acids1-103), DED2 (amino acids 104-180) or the protease domain (a.a. 217-479)of Caspase 8, the corresponding inserts were amplified with PCR using aCaspase 8 cDNA as a template and subsequently cloned into the mammalianexpression vector pcDNA3 (Invitrogen).

ND-Caspase 8 is missing the first 42 amino acids of Caspase 8 and wasgenerating by deleting the first 51 nucleic acids of FLAG-Caspase 8 cDNAclone by taking advantage of a Bgl II site in the Caspase 8 sequence. Asa result of this deletion, the original start site is deleted so thattranslation starts at the methionine residue at position 43.

DN-mTRAF2 is missing the DNA encoding the first 87 amino acids and wasgenerated by using PCR with custom primers to amplify the DNA encodingthe a.a. 88-501 of mTRAF2 and incorporating a start site (i.e.methionine residue) at the N-terminus.

DN-mTRAF5 was constructed similarly to DN-mTRAF2, deleting the first 204amino acids of mTRAF5 clone.

Caspase 8 Mutants

Caspase 8 C360S has the amino acid cysteine (C) at the residue 360replaced by amino acid serine (S). Similar nomenclature applies to themutants Caspase 8 D73A, L74A, and L75A as well as to Caspase 9 C288S andCaspase 7 C186S. To generate Flag-tagged Caspase 8 C360S, Quickchangesite directed mutagenesis kit from Stratagene (La Jolla, Calif.) wasused. Flag-tagged Caspase 8 cDNA was used as a template for mutagenesis.The sequence of the primers was as follows:

    Upper primer:                                                                   5' GTGTTTTTTATTCAGGCTAGTCAGGGGGATAACTACCAGAA 3' (SEQ ID NO:29)                 - Lower primer:                                                              5' TTCTGGTAGTTATCCCCCTGACTAGCCTGAATAAAAAACAC 3' (SEQ ID NO:30)          

Mutagenesis was performed by following the manufacturer's instructions.The sequence of the mutated construct was confirmed by automatedfluorescent dye-terminator sequencing on an ABI 373 sequencing machine.

The same approach was used to generate the mutants in the DEDs ofCaspase 8. The primers used were as following:

    For Caspase 8 D73A:                                                             Upper primer:                                                                 5' TAATAGACTGGCTTTGCTGATTAC 3' (SEQ ID NO:31)                                 Lower primer:                                                                 5' GTAATCAGCAAAGCCAGTCTATTA 3' (SEQ ID NO:32)                                  - For Caspase 8 L74A:                                                        Upper primer:                                                                 5' AATAGACTGGATGCGCTGATTACC 3' (SEQ ID NO:33)                                 Lower primer:                                                                 5' GGTAATCAGCGCATCCAGTCTATT 3' (SEQ ID NO:34)                                  - For Caspase 8 L75A:                                                        Upper primer:                                                                 5' GACTGGATTTGGCGATTACCTACC 3' (SEQ ID NO:35)                                 Lower primer:                                                                 5' GGTAGGTAATCGCCAAATCCAGTC 3' (SEQ ID NO:36)                           

Mutants of Other Caspases

Flag-tagged Caspase 7 C186S and Caspase 9 C288S were constructed in amanner similar to that of the Caspase 8 mutants, using Flag-taggedCaspase 7 or Caspase 9 cDNA as a template for mutagenesis. The sequenceof the primers was as follows:

    For Caspase 7 C186S:                                                            Upper primer:                                                                 5' TCTTCATTCAGGCTAGCCGAGGGACCGAG 3' (SEQ ID NO:37)                            Lower primer:                                                                 5' CTCGGTCCCTCGGCTAGCCTGAATGAAGA 3' (SEQ ID NO:38)                             - For Caspase 9 C288S:                                                       Upper primer:                                                                 5' TTCATCCAGGCCaGTGGTGGGGAGC 3' (SEQ ID NO:39)                                Lower primer:                                                                 5' GCTCCCCACCACTGGCCTGGATGAA 3' (SEQ ID NO:40)                          

Caspase 10 PRO (a.a. 1-191) was constructed by amplifying thecorresponding DNA coding for the desired sequence using PCR and customprimers and subsequent cloning in the pcDNA3 expression vector.

EXAMPLE 1

The following assays were conducted on both 293T cells and on MCF7 cellsto determine the ability of various constructs of Caspase 8 to activatethe NF-κB pathway.

Transfection of 293T Cells

293T cells (1×10⁵) were seeded in each well of a 24 well tissue cultureplate and 24 hours later transfected with either a test vectorcontaining a construct (750 ng) or a control plasmid (750 ng), alongwith an NF-κB/luciferase reporter construct (75 ng) and an RSV promoterdriven β-galactosidase reporter construct (pRcRSV/LacZ) (75 ng) induplicate using a calcium phosphate coprecipitation method. The calciumphosphate coprecipitation method comprises a 2× HEPES solution (8 gNaCl, 1.5 mM Na₂ HPO₄, 6.5 g HEPES, an amount of H₂ O adequate to bringthe total volume of the solution to 500 ml, pH of 7.0, stored at 4° C.)and 2M CaCl₂ stored in aliquots at -20° C. Two and one half (2.5) μl of2M CaCl₂ solution was mixed with the desired DNA construct solutions(dissolved in a buffer containing 10 mM Tris, 1 mM EDTA, and pH=8) andwater in an amount to bring the total volume of the complete solution to20 μl. To this solution, 20 μl of the 2× HEPES solution was addeddropwise and the resulting precipitate was sprinkled over the cells in awell of the 24-well Tissue culture plate (Falcon). Each experiment wasperformed in duplicate.

Transfection of MCF7 Cells

MCF7 cells (1×10⁵) were transfected using 3 μl Superfect (obtained fromQiagen in Valencia, Calif.), following the Manufacturer's instructionswhich accompany the Superfect and as described in Chaudhary et al.,Immunity 7:831-830 (1997).

Luciferase Assays of 293T and MCF7 Cells

Twenty four hours after transfection, cell extracts were prepared usingthe Luciferase Cell Culture Lysis Reagent (Promega, Madison, Wis.;Catalog #E1531). Luciferase assays of the lysates were performed using20 μl of cell extract as described Current Protocols in MolecularBiology, Vol. 1, Chap. 9.7B, John Wiley & Sons, Inc. (1995),incorporated by reference herein, with the exception that 100 μl of a200 μM luciferin solution dissolved in the luciferase assay was addeddirectly to each sample using an automated injector from a Gene-ProbeLuminometer (Berthoid). The cell lysate was diluted 1 to 20 times withPhosphate Buffered Saline (pH=7.4) (Life Technologies, Inc.,Gaithersburg, Md.) and the β-galactosidase activity was measured asdescribed in Molecular Cloning, 2d edition, by Sambrook, Fritsch,Maniatis, Cold Spring Harbor Laboratory Press, 1989, incorporated byreference herein, except that the reaction was stopped by addition of150 μl of 1M Tris pH 8. Absorbance of the final colored product wasmeasured at 415 nm using a Bio-Rad model 3550 microplate reader.Luciferase activity was normalized relative to the β-galactosidaseactivity to control for the difference in the transfection efficiency.The above procedure was repeated with each test vector listed in FIG. 1.

Results

As shown in FIG. 1, expression of Caspase 8 in 293T cells led tosignificant activation of the NF-κB/luciferase reporter construct ascompared to the control vector. An active site mutant of Caspase 8containing a cysteine to serine mutation at the catalytic active site(i.e., Caspase 8 C360S) was as effective as the wild-type Caspase 8 inactivating the NF-κB pathway. This mutant is incapable of induction ofapoptosis when over-expressed in the 293T cells. Caspase 8 alsoactivated NF-κB in the MCF-7 human breast cancer cell line (see FIG. 2).To determine the domains of Caspase 8 that are responsible for NF-κBactivation, the above experiment was repeated with different deletionconstructs of Caspase 8. A construct containing the full-lengthprodomain, (i.e., containing the two DEDs (a.a. 1-180)), was able toactivate NF-κB to a greater extent than the full-length Caspase 8.However, deletion constructs encoding either DED1 (a.a. 1-103) or DED2(a.a. 104-180) failed to do so. Similarly, a construct encoding thefull-length protease domain failed to activate NF-κB. These resultsconfirm that the NF-κB induction by Caspase 8 depend on an intactprodomain containing two DEDs and is independent of the protease domainor the protease/apoptosis inducing activity of Caspase 8.

EXAMPLE 2

Caspase 10 (Mch4 isoform)

In this experiment, the ability of Caspase 10 (Mch4 isoform) to activateNF-κB was tested in 293T cells using the procedure described inExample 1. Caspase 10 is a homolog of Caspase 8 and possesses aprodomain homologous to Caspase 8. Unlike Caspase 8, Caspase 10 ishighly cytotoxic in 293T cells. This experiment therefore tested theability of a proteolytically inactive mutant of Caspase 10 containing acysteine to alanine mutation at the active site, to activate NF-κB. Theresults of this test are shown in FIG. 3. This mutant was highlyeffective in activating NF-κB.

Caspase 10 (FLICE2 isoform)

In order to rule out the possibility that the above ability to activateNF-κB was secondary to the mutation of the active site, the ability ofCaspase 10 (FLICE2 isoform) to activate the NF-κB pathway was testedutilizing the above assay (see FIG. 4). Unlike the Mch4 isoform, FLICE2isoform is not cytotoxic. The FLICE2 isoform was also able toeffectively activate NF-κB. This confirms that the wild-type Caspase 10has the ability to activate NF-κB.

EXAMPLE 3

These experiments tested the ability of several other Caspase familymembers (Caspases 1, 2, 3, 6, 7, 9) to activate the NF-κB pathway in293T cells. The test is procedures followed were identical to those ofExample 1, substituting the particular Caspase family member for thetest vector. The other family members possess a protease domainhomologous to Caspase 8 but do not possess a DEDs-containing prodomain.As can be seen in FIG. 5, none of the other Caspase family members wereable to activate the NF-κB pathway. Caspase 8, however, effectivelyactivated the NF-κB pathway. These results confirm the importance ofDEDs in activation of the NF-κ pathway.

EXAMPLE 4

In this experiment, the ability of MRIT (both MRIT-α1 and MRIT-β1isoforms) to induce the NF-κB pathway in the 293T cells was analyzed.MRIT resembles Caspase 8 in that it possesses a prodomain consisting oftwo homologous DEDs. The procedure followed was as set forth in Example1.

The results are shown in FIG. 6. Compared to the control, MRIT-α1effectively activated the NF-κB pathway. The MRIT-β1 isoform whichcontains the two DEDs was as effective as the full length MRIT-α1 inactivating NF-κB. This further confirms the role of MRIT in activatingNF-κB and localizes this activity to its prodomain.

Following the procedure for MCF7 cell transfection shown in Example 1,it was also determined that MRIT-α1 also activated NF-κB in the MCF7cells.

EXAMPLE 4a

The NF-κB activation mediated by Caspase 8, 10, and MRIT was confinedusing an independent assay based on electrophoretic mobility shift (seeFIG. 7). For this assay, 293T cells were (1×10⁶) transfected with acontrol vector or an expression vector encoding Caspase 8 C360S, Caspase10 C358A, MRIT, CD95/Fas or TNFR1 (5 mg each). After 36 hours, nuclearextracts were prepared as described in Schreiber et al., Rapid detectionof octamer binding proteins with "mini extracts", prepared from a smallnumber of cells, Nucleic Acids Res, 17(15):6419 (1989). Nuclear extracts(2 μl) were incubated for 30 minutes at room temperature with a ³³ Plabeled NF-κB duplex oligonucleotide (Promega, Madison, Wis.; catalogno. E3292) in a buffer containing 10 mM HEPES (pH 7.9); 50 mM KCl, 0.2mM EDTA, 2.5 mM DTT, 10% glycerol and 0.5% NP-40. Protein-DNA complexeswere resolved on a 5% native polyacrylamide and run in 0.5× TBE. Gel wasdried and autoradiographed.

One skilled in the art will appreciate that the electrophoretic mobilityshift assay and the Luciferase-based reporter assay are only two ofnumerous assays which could be utilized to measure activation of NF-κBpathway. Other workable assays include Chloramphenicol Acyl TransferaseAssay (CAT) and Secreatory alkaline phosphatase (SEAP).

EXAMPLE 5

This example illustrates the effect of various molecules on Caspase- andMRIT-induced NF-κB activation. The experiments were conducted asdescribed in Example 1 with the following modifications: the amount oftest plasmid (i.e., Caspase 8 C360S, Caspase 10 C358A, MRIT, or controlvector) used was 100 ng/well, and a second plasmid (i.e., controlvector, crmA, p35, or DN-mTRAF2) was co-transfected along with the testplasmid at 750 ng/well. The results of these tests, set forth in FIG. 8,indicate that MRIT-induced NF-κB activation can be inhibited by crmA,p35, and DN-mTRAF2. However, only DN-mTRAF2 can block Caspase 8- andCaspase 10-induced NF-κB activation. This example indicates that anNF-κB-based functional assay can be utilized as a screening tool foridentifying lead compounds for pharmacological agents capable ofselectively blocking Caspase 8-, Caspase 10-, and/or MRIT-induced NF-κBsignal transduction pathways.

EXAMPLE 6

The object of this test was to determine whether DN-mTRAF5 can blockCaspase 8 -, MRIT- and DR3 -induced NF-κB pathway activation. Theprocedures followed were identical to those of Example 5, using 100ng/well of the first plasmid (i.e., control vector, Caspase 8, MRIT, andDR3) and 750 ng/well of the second plasmid (i.e., control vector,DN-mTRAF2, and DN-mTRAF5). The results are shown in FIG. 9 anddemonstrate that DN-mTRAF5 does not block Caspase 8- and MRIT-inducedNF-κB. Therefore, Caspase 8- and MRIT-induced NF-κB activation does notdepend upon TRAF5.

EXAMPLE 7

This series of experiments was conducted to determine whether mTRAF1 andI-TRAF could block Caspase 8-, MRIT-, and DR3-induced NF-κB activation.The procedures followed were identical to those of Example 5, using 100ng/well of the first plasmid (i.e., control vector, Caspase 8, MRIT, andDR3) and 750 ng/well of the second plasmid (i.e., control vector,mTRAF1, and I-TRAF). The results are shown in FIG. 10 and demonstratethat both mTRAF1 and I-TRAF can block Caspase 8-induced NF-κB, butfailed to effectively block MRIT-induced NF-κB. This example indicatesthat NF-κB-based functional screening assays can be utilized to identifylead compounds for pharmacological agents useful in the selectiveinhibition of Caspase 8-induced NF-κB pathway, while not interferingwith MRIT-induced NF-κB signal transduction pathway.

EXAMPLE 8

The amino acid sequences of various DEDs-containing proteins werecompared in order to identify residues which were highly conserved amongthe various proteins. The multiple sequence alignments of theseDEDs-containing proteins are illustrated in FIG. 11. The conservedresidues appear to play a functional role in the ability ofDEDs-containing proteins to induce NF-κB. Knowledge of this sequenceconservation can be used to develop pharmacological agents with alteredproperties to activate/inhibit the NF-κB pathway using the techniques ofsite-directed mutagenesis and/or structural-based drug design known inthe art.

In order to prove this theory, various deletion and point mutants of theDEDs of Caspase 8 were tested for their ability to block Caspase 8C360S-, MRIT- and DR3-induced NF-κB activation. The procedures followedwere identical to those of Example 5, using 100 ng/well of the firstplasmid (i.e., control vector, Caspase 8 C360S, MRIT, and DR3) and 750ng/well of the second plasmid (i.e., control vector, ND-Caspase 8,Caspase 8 D73A, and Caspase 8 L74A). The results are shown in FIG. 12and demonstrate that the various deletion and point mutants of Caspase 8can block Caspase 8-, MRIT-, and DR3-induced NF-κB. This exampleillustrates that site-directed mutagenesis and structural-based drugdesign can be used to identify lead compounds for a pharmacologicalagent useful in the inhibition of Caspase 8-, MRIT-, and DR3-inducedNF-κB pathway.

EXAMPLE 9

This example illustrates that Caspase 8 plays a role in the activationof the NF-κB pathway mediated by various death receptors belonging tothe TNF receptor family. The procedures followed were identical to thoseof Example 5 using 100 ng/well of the first plasmid (i.e., controlvector, TNFR1, CD95/Fas, DR3, DR4, and DR5) and 750 ng/well of thesecond plasmid (i.e., control vector, Caspase 8 D73A, Caspase 8 L74A,and Caspase 8 L75A). The results are shown in FIG. 13. The mutants D73Aand L74A were unable to activate NF-κB while the mutant L75A partiallyactivated NF-κB. However, all of these mutants of DEDs of Caspase 8blocked activation of the NF-κB pathway mediated by various deathreceptors belonging to the TNF receptor family. Therefore, these andsimilar mutants of the DEDs-containing proteins can serve as leadcompounds for pharmacological agents useful in the treatment of diseasesassociated with dysfunctional NF-κB activation mediated by various deathreceptors of the TNFR family. Furthermore, this example illustrates thatthe structural features of DEDs may be exploited using the techniques ofsite-directed mutagenesis and structural-based drug design to identifylead compounds for pharmacological agents useful in the inhibition ofdeath receptor-induced activation of the NF-κB pathway.

EXAMPLE 10

This test was conducted to test the ability of K13-ORF (a proteinencoded by the Kaposi Sarcoma Associated Herpes Virus) to activate theNF-κB pathway. The procedures followed were identical to those ofExample 5 using 100 ng/well of the first plasmid (i.e., control vectorand K13-ORF) and 750 ng/well of the second plasmid (i.e., controlvector, DN-mTRAF2, I-TRAF, crmA, p35, and DN-TRAF 5). The results areset forth in FIG. 14 and illustrate that K13-ORF activates the NF-κBpathway. Furthermore, K13-ORF mediated NF-κB activation is inhibited byDN-mTRAF2 and I-TRAF, but not by crmA, p35, and DN-TRAF5. An NF-κB-basedfunctional assay can thus be used as a screening tool for identifyinglead compounds for a pharmacological agent capable of selectivelyblocking K13-ORF-induced NF-κB signal transduction pathway for use inthe treatment of disease associated with the dysfunction/activation ofthis pathway.

EXAMPLE 11

Tests were conducted to determine whether MC159L, a DEDs-containingprotein encoded by Molluscum Contagiosum Virus, inhibits the NF-κBpathway. If it does inhibit the NF-κB pathway, MC159L may be responsiblefor the lack of inflammatory response in patients infected with thisvirus. The procedures followed were identical to those of Example 5,using 100 ng/well of the first plasmid (i.e., control vector, Caspase 8C360S, Caspase 10 C358A, and MRIT) and 750 ng/well of the second plasmid(i.e., control vector and MC 159L). The results are shown in FIG. 15 anddemonstrate that MC159L effectively blocks the Caspase 10 C358A- andMRIT-induced NF-κB pathway, but only minimally blocks the Caspase 8C360S-induced NF-κB pathway. This example provides a mechanism by whichMC159L can block the inflammatory response observed among patientsinfected with MCV. Based on these results, the inhibitors of MC159Lmediated inhibition of the NF-κB pathway can be used as lead compoundsfor identifying pharmacological agents useful for the diagnosis andtreatment of MCV infection. Such inhibitors can be readily identifiedusing screening assays for NF-κB activation. Furthermore, based on theseresults, MC159L can be used as a lead compound for identifyingpharmacological agents useful for the treatment of inflammatorydisorders associated with the dysfunction of the NF-κB pathway.

EXAMPLE 12

These tests were conducted to determine whether E8, a DEDs-containingprotein encoded by the Equine Herpes Virus 2, modulates NF-κBactivation. The procedures followed were identical to those of Example5, using 100 ng/well of the first plasmid (i.e., control vector, Caspase8 C360S, Caspase 10 C358A, and MRIT) and 750 ng/well of the secondplasmid (i.e., control vector and E8). The results, shown in FIG. 16,illustrate that by itself E8 only moderately activates NF-κB, butsynergized Caspase 8 C360S-, Caspase 10 C358A-, and MRIT-induced NF-κBactivation. Therefore, an NF-κB-based functional assay can be used as ascreening tool for identifying lead compounds for a pharmacologicalagent capable of enhancing the NF-κB activating abilities of Caspase 8C360S, Caspase 10 C358A, and MRIT. Based on the structure of the DEDs ofE8, lead compounds for identifying pharmacological agents useful formodulating or enhancing NF-κB activation can also be identified.

EXAMPLE 13

These tests were conducted to test the hypothesis that DEDs-containingproteins can function as inhibitors of the NF-κB pathway activated byseveral members of the TNFR family. The procedures followed wereidentical to those of Example 5, using 100 ng/well of the first plasmid(i.e., control vector, TNFR1, Fas/CD95 (GenBank P25445), DR3, DR4,TNFR2, CD40, MTRAF5 and LTBR) and 750 ng/well of the second plasmid(i.e., control vector, Caspase 8 D73A, MC159L and PEA-15). The resultsare set forth in FIG. 17 and demonstrate that Caspase 8 D73A, MC159L andPEA-15 can block activation of NF-κB pathway induced by members of theTNFR family. Furthermore, these results show that there is a differencein the ability of MC159L to inhibit NF-κB activation mediated by thevarious members of the TNFR family. For example, MC159L is a goodinhibitor of NF-κB activation mediated by Fas/CD95, DR4, TNFR2, CD40,MTRAF5 and lymphotoxin-β receptor but is a relatively poor inhibitor ofNF-κB activation mediated by TNFR1 and DR3. The results demonstrate thatthe various DEDs-containing proteins may be used for selectivelymodulating activation of the NF-κB pathway mediated by the variousmembers of the TNF receptor family. The various DEDs-containing proteinsmay also be used as lead compounds for developing pharmacological agentsuseful in selectively modulating activation of the NF-κB pathwaymediated by the various members of the TNF receptor family.

EXAMPLE 14

This experiment was conducted to test the hypothesis thatDEDs-containing proteins can function as an inhibitor of the NF-κBpathway activated by K13-ORF. The procedures followed were identical tothose of Example 5, using 100 ng/well of the first plasmid (i.e.,control vector, or K13-ORF) and 750 ng/well of the second plasmid (i.e.,control vector, Caspase 8 D73A, MC159L and PEA-15). The results aregiven in FIG. 18 and indicate that Caspase 8 D73A, MC159L and PEA-15 canblock activation of NF-κB pathway induced by K13-ORF. The variousDEDs-containing proteins may be used as lead compounds for developingpharmacological agents useful in the diagnosis or treatment of diseaseassociated with dysfunction of the NF-κB signal transduction pathwaymediated by K13-ORF.

EXAMPLE 15

These tests were conducted to determine whether DEDs-containing proteinscan function as an inhibitor of the NF-κB pathway activated by RIP. Theprocedures followed were identical to those of Example 5, using 100ng/well of the first plasmid (i.e., control vector, or RIP) and 750ng/well of the second plasmid (i.e., control vector, Caspase 8 D73A,Caspase 8 L75A, MC159L and PEA-15). The results are illustrated in FIG.19. Caspase 8 D73A, Caspase 8 L75A, MC159L and PEA-15 can blockactivation of the NF-κB pathway induced by RIP. Thus, variousDEDs-containing proteins may be used for modulating activation of theNF-κB pathway mediated by RIP. The various DEDs-containing proteins mayalso be used as lead compounds for developing pharmacological agentsuseful in modulating activation of NF-κB pathway mediated by RIP.

EXAMPLE 16

This experiment was conducted to test the hypothesis that Caspase 3 canfunction as an inhibitor of the NF-κB pathway activated by Caspase 8,MRIT and DR3. The procedures followed were identical to those of Example5, using 100 ng/well of the first plasmid (i.e., control vector, Caspase8 C360S, MRIT and DR3) and 750 ng/well of the second plasmid (i.e.,control vector, Caspase 3). The results are shown in FIG. 20 anddemonstrate that Caspase 3 can block activation of the NF-κB pathwayinduced by Caspase 8 C360S, MRIT and DR3. Thus, Caspase 3 may be usedfor selectively inhibiting activation of the NF-κB pathway mediated byCaspase 8, MIT, DR3, as well as NF-κB pathway induced by other TNFRfamily. Caspase 3 may also be used as a lead compound for developingpharmacological agents useful in inhibiting the activation of theNF-KB-KB pathway mediated by Caspase 8, MRIT and TNFR family members.

EXAMPLE 17

These tests were conducted to determine whether A20, mTRAF1, Caspase 7C186S and Caspase 9 C288S can function as inhibitors of the NF-κBpathway activated by Caspase 8 C360S, Caspase 10 C358A, MRIT andK13-ORF. The procedures followed were identical to those of Example 5,using 100 ng/well of the first plasmid (i.e., control vector, Caspase 8C360S, Caspase 10 C358A, MRIT and K13-ORF) and 750 ng/well of the secondplasmid (i.e., control vector, A20, Caspase 7 C186S and Caspase 9C288S). The results, shown in FIG. 21, indicate that A20, Caspase 7 C186S and Caspase 9 C288S blocks the NF-κB pathway mediated by Caspase 8C360S, Caspase 10 C358A, MRIT and K13-ORF. Thus, A20, Caspase 7 C186S,and Caspase 9 C288S may be used for inhibiting activation of the NF-κBpathway mediated by Caspase 8, Caspase 10, MRIT and K13-ORF.Furthermore, A20, Caspase 7 C186S, and Caspase 9 C288S may serve as leadcompounds for developing pharmacological agents useful in inhibitingactivation of the NF-κB pathway mediated by Caspase 8, Caspase 10, MRITand K13-ORF.

EXAMPLE 18

This experiment was conducted to test whether non-DEDs-containingCaspases such as Caspase 7 C186S or Caspase 9 C288S can function asinhibitors of the NF-κB pathway activated by members of the TNFR family(i.e. TNFR1, Fas/CD95, DR3 and DR4). The procedures followed wereidentical to those of Example 5, using 100 ng/well of the first plasmid(i.e., control vector, TNFR1, Fas/CD95, DR3 and DR4) and 750 ng/well ofthe second plasmid (i.e., control vector, Caspase 7 C186S and Caspase 9C288S). The results are set forth in FIG. 22 and demonstrate thatCaspase 7 C186S and Caspase 9 C288S can block NF-κB pathway mediated bymembers of the TNFR family leading to the conclusion that othernon-DEDs-containing Caspases may also be used to inhibit suchTNFR-induced pathways. Furthermore, Caspase 7 C 186S, and Caspase 9C288S may serve as lead compounds for developing pharmacological agentsuseful in inhibiting activation of NF-κB pathway mediated by the variousmembers of the TNFR family.

EXAMPLE 19

These tests were conducted to determine whether a dominant negativemutant of NIK can function as an inhibitor of the NF-κB pathwayactivated by Caspase 8, Caspase 10 and MRIT. The procedures followedwere identical to those of Example 5, using 100 ng/well of the firstplasmid (i.e., control vector, Caspase 8 C360S, Caspase 10 C358A andMRIT) and 750 ng/well of the second plasmid (i.e., control vector,DN-NIK). The results, set forth in FIG. 23, demonstrate that DN-NIK canblock activation of the NF-κB pathway induced by Caspase 8 C360S,Caspase 10 C358A and MRIT. Thus, DN-NIK may be used for inhibitingactivation of the NF-κB pathway mediated by Caspase 8, Caspase 10 andMRIT. DN-NIK may also be used a lead compound for developingpharmacological agents useful in inhibiting activation of NF-κB pathwaymediated by Caspase 8, Caspase 10 and MRIT.

EXAMPLE 20

In this experiment, the ability of various Caspases and of MRIT toactivate the JNK pathway is demonstrated. The luciferase based assay formeasuring the activation of the JNK pathway was conducted using thec-Jun PathDetect™ Reporting System (Stratagene, La Jolla, Calif.,catalog no. 219000). Briefly, 293EBNA cells (1×10⁵) (Invitrogen) weretransfected with 750 ng/well of a test plasmid (i.e., control vector,Caspase 8 C360S, Caspase 10 C358A, MRIT, Caspase 8 PRO (a.a. 1-180),Caspase 10 PRO (a.a. 1-191), MRIT-β1, CD40) along with pFA-Jun (50 ng)pFR-Luc (500 ng), and a pRcRSV/lacZ plasmid in duplicate in each well ofa 24 well tissue culture plate using the calcium phosphateco-precipitation method described in Example 1. Luciferase andβ-galactosidase activities were measured from cell extracts between24-40 hours after transfection as described above for NF-κB assay.β-galactosidase activity was used to normalize the luciferase activityto control for variations in the transfection efficiency.

The results of this experiments are set forth in FIG. 24 and demonstratethat Caspase 8, Caspase 10, and MRIT can activate the JNK pathway andthat this activity is localized to their respective pro-domainsconsisting of the two DEDs.

EXAMPLE 21

In this experiment the ability of various Caspases and MRIT to activatethe JNK pathway was demonstrated using a JNK activation assay based onthe phosphorylation of c-jun. 293 EBNA cells (2×10⁶ cells) weretransfected with 5 μg of each of the test plasmid (i.e., control vector,Caspase 8 C360S, Caspase 10 C358A, MRIT, and CD40) and a reporterplasmid encoding HA-tagged Green Fluorescent protein (GFP-HA).Thirty-six hour post-transfection the cells were lysed and an assay forphosphorylated c-jun was performed using the Non-radioactive SAPK/JNKAssay Kit (New England BioLabs, catalog no. 9810) and following themanufacturer's instructions.

The results of these experiments are set forth in FIG. 25. Caspase 8,Caspase 10, and MRIT activate the JNK pathway. This activity isindependent of the protease activity of Caspases 8 and 10. This exampleillustrates that the above assay, as well as similar JNKactivation-based functional assays known to one skilled in the art, canbe used as a screening tool for identifying lead compounds forpharmacological agents capable of selectively modulating Caspase 8-,Caspase 10-, and/or MRIT-induced JNK signal transduction pathway.

EXAMPLE 22

These tests were conducted to determine whether various agents werecapable of modulating Caspase 8-, Caspase 10-, and MRIT-inducedactivation of the JNK pathway. The procedure followed was as describedin Example 20 except that 750 ng/well of the second plasmid (i.e.,control vector, DN-mTRAF2, I-TRAF, Caspase 8 D73A, and Caspase 8 L75A)was used along with 100 ng/well of the test plasmid (i.e., controlvector, Caspase 8 C360S, Caspase 10 C358A, and MRIT).

The results of this experiment are set forth in FIG. 26 and demonstratethat DN-mTRAF2, I-TRAF, Caspase 8 D73A, and Caspase 8 L75A can blockCaspase 8-, Caspase 10-, and MRIT-induced activation of the JNK pathway.This example indicates that a JNK activation-based functional assay canbe used as a screening tool for identifying lead compounds forpharmacological agents capable of blocking Caspase 8-, 10-, and/orMRIT-induced JNK signal transduction pathway.

EXAMPLE 23

The purpose of this experiment was to determine the ability of a Caspase8 mutant (Caspase 8 D73A) to inhibit TNF receptors-induced activation ofthe JNK pathway. The procedure followed was as described in Example 20except that 750 ng/well of the second plasmid (i.e., control vector andCaspase 8 D73A) was used along with 100 ng/well of the test plasmid(i.e., control vector, TNFR1, CD95/FAS, DR3, DR4, and CD40). The resultsof this experiment are set forth in FIG. 27. Caspase 8 D73A can blockTNF receptors-induced activation of the JNK pathway. This example showsthat a JNK activation-based functional assay can be used as a screeningtool for identifying lead compounds for a pharmacological agent capableof selectively blocking TNF receptors-induced JNK signal transductionpathway. Furthermore, this example illustrates that the structuralfeatures of DEDs-containing proteins may be exploited using thetechniques of site directed mutagenesis and structural-based drug designto identify lead compounds for a pharmacological agent useful in theinhibition of TNF receptors-induced activation of the JNK pathway.

EXAMPLE 24

These experiments were conducted to determine whether Caspase 8 caninteract with TRAF1 and TRAF2 proteins using the followingco-expression-immunoprecipitation assay.

For studying in vivo interaction, 2×10⁶ 293T cells were plated in a 100mm plate. Eighteen to 24 hours later, the cells were co-transfected with5 μg/plate of each of the first epitope-tagged constructs (i.e., controlvector and myc-Caspase 8) along with 5 μg/plate of either HA-epitopetagged mTRAF1 or FLAG-epitope tagged mTRAF2, in combination with 1 μg ofa Green Fluorescent Protein (GFP) encoding plasmid (pEGFP-C1)(Clontech). The co-transfection was achieved by the calcium phosphateco-precipitation method which comprises a 2× HEPES solution (8 g NaCl,1.5 mM Na₂ HPO₄, 6.5 g HEPES, an amount of H₂ O to bring the totalsolution volume to 500 ml, pH of 7.0, stored at 4° C.) and 2M CaCl₂stored in aliquots at -20° C. Sixty-one μl of 2M CaCl₂ solution wasmixed with the desired DNA construct solutions (dissolved in a buffercontaining 10 mM Tris and 1 mM EDTA) and water in an amount to bring thetotal volume of the complete solution to 500 μl. To this solution, 500μl of the 2× HEPES solution was added dropwise with shaking and theresulting precipitate was sprinkled over the cells in each tissueculture plate (Falcon). Eighteen to 36 hours post-transfection, thecells were examined under a fluorescent microscope to ensure equaltransfection efficiency as determined by the expression of the GFP.Eighteen to 36 hours post-transfection cells were lysed in 1 ml of alysis buffer containing 0.1% Triton-X 100, 20 mM sodium phosphate (pH7.4), 150 mM NaCl and 1 EDTA free protease inhibitor tablet per 10 ml(Boehringer Mannheim). For immunoprecipitation, the cell lysate (500 μl)was incubated for 1 hour at 4° C. with 10 μl of myc beads or controlbeads precoated with 2% BSA. The beads were washed twice with lysisbuffer, twice with a wash buffer (0.1% Triton-X 100, 20 mM sodiumphosphate (pH 7.4), 500 mM NaCl), and again with lysis buffer. Boundproteins were eluted by boiling for 3 minutes in SDS-loading buffer,separated by SDS-PAGE, and transferred to a nitrocellulose membranefollowed by a western blot analysis. For the western blot analysis, thenitrocellulose membrane was pre-blocked with 5% casein in TBS with 0.05%Tween 20. Coimmunoprecipitating TRAF1 and TRAF2 proteins were detectedby western blot analysis using rabbit polyclonal antibodies against theHA or the FLAG™ epitope tags respectively. Horse-radish-peroxidaseconjugated donkey anti-rabbit secondary antibody was obtained fromPierce (Catalog #31458). Incubation with primary and secondaryantibodies was carried out in 2% casein. Between each incubation, themembrane was washed three to four times with TBS containing 0.05% Tween.The blot was developed using the Supersignal ULTRA (Pierce)Chemiluminescent Detection System following the manufacturer'sinstructions.

The results of these experiments are given in FIG. 28. Caspase 8directly interacts with both TRAF1 and TRAF 2 proteins when co-expressedwith TRAF1 or TRAF2 in mammalian cells. This example demonstrates thatan assay based on interaction between Caspase 8 and TRAF1 or TRAF2 canbe used as a screening tool for identifying lead compounds forpharmacological agents capable of selectively blocking Caspase 8 andTRAF1 or TRAF2 interaction for the purpose of blocking Caspase 8-TRAFssignal transduction pathway.

EXAMPLE 25

This experiment was conducted to determine whether Caspase 8 interactswith the TRAF3 protein. The co-expression-immunoprecipitation assay wassimilar to that described in Example 24 except the first expressionvector consisted of FLAG-tagged Caspase 8 C360S and the secondexpression vector consisted of HA-tagged TRAF3. FLAG-tagged proteinswere immunoprecipitated using the FLAG beads or control beads, andco-immunoprecipitating HA-TRAF3 was detected by western blot analysisusing a rabbit polyclonal antibody against the HA tag.

The results of this experiment are shown in FIG. 29 and indicate thatCaspase 8 directly interacts with the TRAF3 protein when they areco-expressed in mammalian cells. Caspase 8 did not interact with GFP-HA,thereby confirming the specificity of its interaction with TRAF3. Thisexample also illustrates that an assay based on the interaction betweenCaspase 8 and TRAF3 can be used as a screening tool for identifying leadcompounds for pharmacological agents capable of selectively blockingCaspase 8 and TRAF3 interaction for the purpose of blocking Caspase8-TRAF3 signal transduction pathway.

EXAMPLE 26

The purpose of these tests was to determine whether Caspase 8 interactswith the TRAF5 protein. The co-expression-immunoprecipitation assay wassimilar to that described in Example 24 except the first expressionvector consisted of myc-tagged Caspase 8 C360S and the second expressionvector consisted of FLAG-tagged TRAF5 or FLAG-tagged DN-TRAF5.FLAG-tagged proteins were immunoprecipitated using FLAG beads or controlbeads. Co-immunoprecipitating myc-tagged Caspase 8 was detected bywestern blot analysis using a rabbit polyclonal antibody against the myctag.

The results of this experiment are set forth in FIG. 30. Caspase 8directly interacts with TRAF5 and DN-TRAF5 proteins when co-expressedwith TRAF5 or DN-TRAF5 proteins in mammalian cells. This example showsthat an assay based on interaction between Caspase 8 and TRAF5 orDN-TRAF5 can be used as a screening tool for identifying lead compoundsfor pharmacological agents capable of selectively blocking Caspase 8 andTRAF5 interaction for the purpose of blocking Caspase 8-TRAF5 signaltransduction pathway.

EXAMPLE 27

This experiment was conducted to determine which domains of Caspase 8and MRIT interact with the TRAF1 protein. The procedure was similar tothe co-expression-immunoprecipitation assay described in Example 24except the first expression vector consisted of FLAG-tagged proteasedomain of Caspase 8 (a.a. 217-479), FLAG-tagged Caspase 8 prodomain(a.a. 1-180), or MRIT-β1 (a.a. 1-221), and the second expression vectorconsisted of HA-tagged mTRAF1. FLAG-tagged proteins wereimmunoprecipitated using FLAG beads or control beads.Co-immunoprecipitating HA-TRAF1 was detected by Western Blot analysisusing a rabbit polyclonal antibody against the HA tag.

The results of this experiment are illustrated in FIG. 31 and show thatthe Caspase 8 protease domain, Caspase 8 prodomain, and MRIT-β1 isoform(containing its prodomain) directly interact with the TRAF1 protein whenco-expressed with it in mammalian cells. Essentially similar resultswere obtained using mTRAF2 protein instead of mTRAF1 (see FIG. 29).Thus, an assay based on interaction between the Caspase 8 proteasedomain, the Caspase 8 prodomain, and the MRIT-β1 isoform, and the TRAFfamily of proteins can be used as a screening tool for identifying leadcompounds for pharmacological agents capable of selectively blocking theinteractions between the Caspase 8 protease domain, Caspase 8 prodomain,and MRIT-β1 isoform, and TRAF proteins for the purpose of blockingCaspase 8-TRAF and MRIT-TRAF signal transduction pathway.

EXAMPLE 28

This experiment was conducted to determine whether FADD can influencethe interaction between the Caspase 8 prodomain and TRAF proteins. Theprocedure was similar to that described in Example 24 except theco-transfection was performed using expression vectors encodingFLAG-tagged Caspase 8 prodomain (a.a. 1- 180) and either HA-taggedmTRAF1 or HA-tagged mTRAF2 in the absence or presence of AU1-tagged FADDas shown in FIG. 33. FLAG-tagged proteins were immunoprecipitated usingFLAG beads or control beads, and co-immunoprecipitating HA-mTRAF1/mTRAF2were detected by western blot analysis using a rabbit polyclonalantibody against the HA tag.

The results of this experiment demonstrate that co-expression of FADDleads to a significant decrease in the amount co-precipitating mTRAF1 ormTRAF2. Therefore, an assay based on the interaction between the Caspase8 prodomain and TRAFs can be used as a screening tool for identifyinglead compounds for pharmacological agents capable of blocking Caspase 8prodomain and TRAFs interactions for the purpose of blocking Caspase8-TRAFs signal transduction pathway.

EXAMPLE 29

As demonstrated in FIG. 27, the protease domain (ICE-homology domain orCaspase domain) of Caspase 8 can interact with TRAF proteins. Thisexperiment was conducted to determine whether interaction with TRAFproteins is a general property of the protease domain which cantherefore be extended to non-DEDs-containing Caspases as well.Therefore, this experiment examined whether Caspase 7, anon-DEDs-containing Caspase, can interact with TRAF1 and NIK proteins.The co-expression-immunoprecipitation assay utilized was similar to thatdescribed in Example 24 except the first expression vector consisted ofFLAG-tagged Caspase 7 C186S and the second expression vector consistedof HA-tagged mTRAF1 or HA-tagged NIK. FLAG-tagged proteins wereimmunoprecipitated using FLAG beads or control beads, andco-immunoprecipitating HA-mTRAF1 or HA-NIK was detected by western blotanalysis using a rabbit polyclonal antibody against the HA tag.

The results of this experiment are shown in FIG. 34 and demonstrate thatCaspase 7 directly interacts with TRAF1 and NIK proteins whenco-expressed with TRAF1 or NIK in mammalian cells. This example alsoillustrates that an assay based on the interactions between Caspase 7and TRAF1 or NIK can be used as a screening tool for identifying leadcompounds for pharmacological agents capable of inhibiting theinteractions between Caspase 7 and TRAF1, or Caspase 7 and NIK, for thepurpose of diagnosis and treatment of diseases associated with thedysfunction of the Caspase 7-TRAF1 and Caspase 7-NIK signal transductionpathway.

EXAMPLE 30

In Example 27, it was demonstrated that two DEDs-containing proteins,the Caspase 8 prodomain and the MRIT-β1 isoform (containing itsprodomain) can interact with the mTRAF1 protein. This experiment wasconducted to determine whether DEDs-containing proteins in general caninteract with the TRAF family of proteins using theco-expression-immunoprecipitation assay. The procedure was similar tothat described in Example 24 except the first expression vectorconsisted of FLAG-tagged K13-ORF, MC159L, MRIT-β1, or PEA-15 and thesecond expression vector consisted of HA-tagged mTRAF2. FLAG-taggedproteins were immunoprecipitated using FLAG beads.Co-immunoprecipitating HA-mTRAF2 was detected by western blot analysisusing a rabbit polyclonal antibody against the HA tag.

The results of this experiment are shown in FIG. 35 and demonstrate thatDEDs-containing proteins interact with TRAF2. This example combined withExample 27 also illustrates that interaction with TRAF proteins is ageneral property of DEDs-containing proteins. An assay based on theseinteractions between DEDs-containing proteins and the TRAF family ofproteins can therefore be used as a screening tool for identifying leadcompounds for pharmacological agents capable of blocking DEDs-TRAFsinteraction for the purpose of inhibiting DEDs-TRAFs signal transductionpathway.

EXAMPLE 31

This experiment was conducted to determine whether various Caspases andDEDs-containing proteins interact with NIK proteins, a serine-threoninekinase involved in the activation it he NF-κB pathway. Theco-expression-immunoprecipitation assay utilized was similar to thatdescribed in Example 24 except the first expression vector consisted ofFLAG-tagged Caspase 8 C360S, Caspase 8 protease domain (a.a. 217-479),Caspase 10, MRIT, Caspase 7, K13-ORF, or PEA-15, and the secondexpression vector consisted of HA-tagged NIK. FLAG-tagged proteins wereimmunoprecipitated using FLAG beads or control beads, andco-immunoprecipitating HA-NIK was detected by western blot analysisusing a rabbit polyclonal antibody against the HA tag.

The results of this experiment are shown in FIG. 36 and demonstrate thatCaspase 8, Caspase 8 protease domain (a.a. 217-479), Caspase 10, MRIT,Caspase 7, K13-ORF, and PEA-15 directly interact with NIK proteins whenco-expressed with NIK in mammalian cells. This example shows that DEDs-and protease domain-containing proteins can in general interact withNIK. An assay based on this interaction can be used as a screening toolfor identifying lead compounds for pharmacological agents capable ofblocking DEDs or protease domain-containing proteins with NIKinteraction for the purpose of inhibiting DEDs-containing proteins-NIKand protease domain-containing proteins-NIK signal transduction pathway.

EXAMPLE 32

This experiment was conducted to determine whether TRAF proteinsinfluence the interactions between Caspase 8 and NIK. The procedure wassimilar to that described in Example 24 except the expression vectorsencoding FLAG-tagged Caspase 8 and HA-tagged NIK were co-transfected inthe absence or presence of HA-mTRAF1 as shown in FIG. 37. FLAG-taggedproteins were immunoprecipitated using FLAG beads or control beads.Co-immunoprecipitating HA-NIK and/or HA-mTRAF1 were detected by westernblot analysis using a rabbit polyclonal antibody against the HA tag. Asimilar procedure was followed using mTRAF2 in place of mTRAF1.

The results of this experiment are set forth in FIGS. 37 and 38 anddemonstrate that co-expression of TRAF proteins leads to a significantincrease in the amount of co-precipitating NIK. Therefore, TRAF proteinsmay be useful as lead compounds for identifying pharmacological agentscapable of modulating Caspases-NIK interactions. This example alsoillustrates that an assay based on the interactions between Caspase 8and NIK can be used as a screening tool for identifying lead compoundsfor pharmacological agents capable of modulating Caspase 8 and NIKinteractions for the purpose of modulating Caspase 8-NIK signaltransduction pathway.

EXAMPLE 33

This experiment was conducted to determine whether various Caspases andDEDs-containing proteins can interact with RIP, another serine-threoninekinase involved in the inactivation of the NF-κB pathway. Theco-expression-immunoprecipitation assay utilized was similar to thatdescribed in Example 24 except the first expression vector consisted ofFLAG-tagged Caspase 8 C360S, Caspase 10 C358A, MRIT, Caspase 8 prodomain(a.a. 1-180), MRIT-β1 isoform (containing its prodomain), K13-ORF,MC159L, or Caspase 7 C186S, and the second expression vector consistedof HA-tagged RIP. FLAG-tagged proteins were immunoprecipitated usingFLAG beads or control beads. Co-immunoprecipitating HA-RIP was detectedby Western Blot analysis using a rabbit polyclonal antibody against theHA tag.

The results of this experiment are shown in FIG. 39. Caspase 8 C360S,Caspase 10 C358A, MRIT, Caspase 8 prodomain (a.a. 1-180), MRIT-β1isoform (containing its prodomain), K13-ORF, MC159L, and Caspase 7 C186Sdirectly interact with RIP protein when co-expressed with RIP inmammalian cells. This example indicates that Caspases andDEDs-containing proteins can, in general, interact with RIP. An assaybased on interactions between Caspases and DEDs-containing proteins withRIP can be used as a screening tool for identifying lead compounds forpharmacological agents capable of blocking interactions between Caspasesand DEDs-containing proteins with RIP for the purpose of inhibitingDEDs-containing proteins-RIP and Caspases-RIP signal transductionpathways.

EXAMPLE 34

This experiment was conducted to determine whether Caspase 8 caninteract with IKK1 (or IKKα) and IKK2 (or IKKβ) proteins. IKK1 and IKK2,like RIP and NIK, are serine-threonine protein kinases involved in theactivation of NF-κB pathway. The co-immunoprecipitation assay followedwas similar to that described in Example 24 except the first expressionvector consisted of myc-tagged Caspase 8 C360S, and the secondexpression vector consisted of FLAG-tagged IKK1 or IKK2 respectively.FLAG-tagged proteins were immunoprecipitated using FLAG beads or controlbeads. Co-immunoprecipitating myc-Caspase 8 was detected by western blotanalysis using a rabbit polyclonal antibody against the myc tag.

The results of this experiment are shown in FIG. 40 and demonstrate thatCaspase 8 directly interacts with IKK1 and IKK2 proteins whenco-expressed with IKK1 or IKK2 in mammalian cells. Furthermore, combinedwith Examples 29, 31, and 33, this example confirms that Caspases andDEDs-containing proteins can interact with serine-threonine proteinkinases. An assay based on interaction between Caspases andDEDs-containing proteins with IKK1 or IKK2 can be used as a screeningtool for identifying lead compounds for pharmacological agents capableof modulating interactions between Caspases (and/or DEDs-containingproteins) and IKK1 or IKK2 for the purpose of diagnosis and treatment ofdiseases associated with the dysfunction of DEDs-containingproteins-IKK1/IKK2 and Caspases-IKK1/IKK2 signal transduction pathways.Similar experiments confirmed that IKK1 also interacts with MRIT-α1.

EXAMPLE 35

This experiment was conducted to test whether Caspase 8 interacts withTRAF proteins in a cell-free system using co-immunoprecipitation ofbacterially expressed proteins. Myc-Caspase 8, mTRAF2-FLAG, mTRAF1-HA ora control vector were cloned in pET28 expression vectors. The expressionplasmids encoding the above proteins were transformed in to the BL21(DE3)pLysS host cells. Expression of the target proteins were inducedusing IPTG and following the manufacturer's instructions (Novagen,Madison, Wis.). After induction for 3 hours, the host cells werepelleted by centrifugation at 4° C. at 3000 g for 10 minutes. The pelletwas resuspended in a buffer (20 mM sodium phosphate, 500 mM NaCl, and 1EDTA-free mini protease inhibitor tablet (Boehringer Mannheim) per 10 mlof buffer) with an amount of buffer which equaled 1/10 of the originalculture volume. Cells were lysed by three cycles of freeze-thawing andsubsequently sonicated (10 pulses of 5 second each at low energy withoutput to dial at level 4) at 4° C. using a Branson Sonifier Model 250.The sonicated samples were centrifuged at 10000 g at 4° C., and thesupernatant containing the soluble fraction was collected. Five hundredμl of supernatant from cultures expressing mTRAF2-FLAG or mTRAF1-HA wereincubated for 1 hour at 4° C. with an equal volume of supernatant fromcultures expressing myc-Caspase 8 or control vectors.Immunoprecipitation was carried out with myc beads as previouslydescribed, and western blot analysis was performed using rabbitpolyclonal antibodies against the HA or FLAG tags as describedpreviously.

The results of this experiment are shown in FIG. 41 and indicate thatCaspase 8 can directly interact with TRAF1 or TRAF2 proteins incell-free systems. This example demonstrate that a cell-free bindingassay based on interaction between Caspase 8 and TRAF1 or TRAF2 can alsobe used as a screening tool for identifying lead compounds forpharmacological agents capable of selectively blocking Caspase 8 andTRAF1 or TRAF2 interaction for the purpose of blocking Caspase 8-TRAFssignal transduction pathways.

EXAMPLE 36

This experiment was conducted to demonstrate that K13-ORF, aDEDs-containing protein, directly interacts with TRAF proteins in theyeast two-hybrid system. For this purpose, K13-ORF was cloned in the DNAbinding domain vector pLexA, and mTRAF1 or mTRAF2 were cloned in theactivation domain vector pB42AD. The yeast two-hybrid interaction assaywas performed as described in the manual accompanying the MATCHMAKERLexA Two-Hybrid System (Clontech, Palo Alto, Calif.).

The results observed in this experiment indicated that K13-ORF directlyinteracts with either mTRAF1 or mTRAF2 in the yeast two-hybridinteraction assay. The results further show that a yeast two-hybridinteraction assay between K13-ORF and TRAF1 can be used as a screeningtool for identifying lead compounds for pharmacological agents capableof blocking K13 -ORF and TRAF1 or TRAF2 interaction for the purpose ofinhibiting K13-ORF-TRAFs signal transduction pathways.

EXAMPLE 37

The purpose of this experiment was to determine the role of TRAFproteins in the mediation of cell death induced by various Caspases. Forthis purpose, 293T cells (1×10⁵) were seeded in each well of a 24 welltissue culture plate. Twenty-four hours later, the cells weretransfected with 250 ng/well of a first plasmid (control vector, Caspase8, Caspase 8 C360S, or Caspase 8 prodomain (a.a. 1-180) along with 250ng/well of a second plasmid (control vector, mTRAF2, or mTRAF2+crmA)using the calcium phosphate co-precipitation method described inExample 1. An RSV/LacZ reporter plasmid (100 ng/well) encoding theβ-galactosidase protein was co-transfected into all the wells. Twentyfour hours after transfection, the cells were fixed and stained withX-gal. The percentage of apoptotic cells was determined as described inChaudhary et al., Immunity, 7:821-830 (1997).

The results of this experiment are shown in FIG. 42 and indicate thatmTRAF2 has slight pro-apoptotic ability when expressed alone inmammalian cells. However, in the presence of the various Caspase 8constructs, an increase in the number of apoptotic cells is observable,indicating that TRAF2 and Caspase 8 cooperate with each other to inducecell death. Essentially similar results were obtained when mTRAF2 wasco-expressed with MRIT. These results demonstrate that agentsinterfering with Caspase-TRAF or MRIT-TRAF interactions may be useful aslead compounds for identifying pharmacological agents useful for thetreatment of diseases resulting from the dysfunction of apoptoticpathways.

EXAMPLE 38

This experiment was conducted to determine the role of activation ofNF-κB and JNK pathways in the mediation of cell death induced by variousCaspases. For this purpose, selective inhibitors of the NF-κB pathway(i.e., IκB-ΔN) and of the JNK pathway (i.e., JIP) were used to blockcell death mediated by Caspase 10 (Mch4 isoform) and Caspase 9. Theprocedure used was similar to that described in Example 37 except that293T cells were transfected with 100 ng/well of the first plasmid (i.e.,control vector, Caspase 10 (Mch4 isoform), and Caspase 9), along with750 ng/well the second plasmid (i.e., control vector, IκB-ΔN, DN-IkappaBand JIP).

The results of this experiment are set forth in FIG. 43 and indicatethat Caspase 10 and Caspase 9 induced-apoptosis can be blocked by IκB-ΔNand JIP. Thus, activation of the NF-κB and JNK pathways plays animportant role in the mediation of cell death induced by Caspases.Agents interfering with activation of NF-κB and JNK pathways may beuseful as lead compounds for identifying pharmacological agents for thetreatment of diseases resulting from the dysfunction of apoptoticpathways.

EXAMPLE 39

This experiment was conducted to determine the role of TRAF proteins inthe mediation of cell death induced by various Caspases. The procedureused was similar to that described in Example 37 except that the cellswere transfected with 100 ng/well of the first plasmid (control vector,Caspase 10, Caspase 9 or Caspase 7) along with 750 ng/well of the secondplasmid (control vector, mTRAF1, mTRAF2, TRAF3, mTRAF5 and TRAF6) usingthe calcium phosphate co-precipitation method described in Example 1.The results of this experiment are shown in FIG. 44 and indicate mTRAF2and TRAF5 have slight pro-apoptotic ability when expressed alone inmammalian cells. However, in the presence of the various Caspaseconstructs, an increase in the number of apoptotic cells is seenindicating that Caspases and TRAFs cooperate with each other to inducecell death. These results demonstrate that agents interfering withCaspase-TRAF interaction may be useful as lead compounds for identifyingpharmacological agents for the treatment of diseases resulting from thedysfunction of apoptotic pathways.

In the course of development of the present invention, novel signaltransduction pathways important for the regulation of apoptosis andimmune and inflammatory responses have been discovered. The inventionmakes use of this knowledge to provide methods for the identification,development and use of therapeutic agents (or lead compounds therefor)which intervene at specific points in these pathways. The following arecertain specific therapeutic applications within the ambit of theinvention.

It has been demonstrated that DEDs-containing Caspases and Caspasehomologs (e.g., MRIT-α1) can directly interact with TRAF family ofadaptor proteins and activate the NF-κB pathway, and that thisinteraction has functional significance in the activation of the NF-κBpathway by the members of the TNFR family. Several dominant negativeinhibitors of this interaction have been identified which can block theactivation of NF-κB pathway by the TNFR family members. Additionalinhibitors of this interaction can thus be readily developed based onthe knowledge that conserved residues among the differentDEDs-containing proteins are critical for this activity. For example, ithas been found that mutation of a single amino acid at the conservedresidues 73, 74 or 75 results in the partial or complete loss of NF-κBactivation by Caspase 8, and that these mutants can block NF-κBactivation by wild-type Caspase 8 and TNFR family members. Several otheramino acids are highly conserved among the various DEDs-containingproteins and may be readily tested using site-directed mutagenesis. Suchmutants may be useful therapeutic agents (or lead compounds) forcontrolling cell death and inflammation resulting from the activation ofthe NF-κB pathway. One scenario would be the use of gene therapy withthese agents for the primary or secondary prevention of coronary arterydisease. As mentioned above, activation of NF-κB pathway has beenimplicated in the pathogenesis of atherosclerosis. With the rapidadvances in the gene therapy technology, it is possible to deliver sucha mutated gene to the endothelial cells lining the coronary arteriesusing either liposomes or viral vectors at the time of coronaryangioplasty. Similarly, such dominant negative inhibitors may havepotential applications in the gene therapy for rheumatoid arthritis,cancer and AIDS.

Another area of potential therapeutic application(s) in the area of cellpermeable peptide analogs. These analogs can be easily synthesized basedon the knowledge of the primary sequence of the interacting domains. Forexample, it is possible to synthesize small peptides or polypeptidescorresponding to the region (s) that are conserved among the variousDEDs-containing proteins. Such small peptides are either intrinsicallycell permeable or made so by the attachment of a side chain. A goodexample of this approach is provided by z-VAD-fink, a cell permeablesmall peptide inhibitor of the protease activity of several Caspases.Similar analogs are currently under development or in clinical trialsfor the treatment of liver failure and neurological disorders resultingfrom overactivity of caspases.

Development of small molecule inhibitors represent another potentialarea of drug discovery based on the present invention. Using thetechniques of combinatorial chemistry, small molecules can besynthesized which may bind to the interacting domains of Caspases, TRAFsor serine-threonine protein kinases. Such small molecules could bereadily screened for their ability to inhibit Caspase induced NF-κB bythe methods hereof.

It has also been discovered that interaction of Caspase 8, 10 andMRIT-α1 also leads to the activation of JNK pathway. Therefore, theagents identified herein may find usefulness in the treatment of diseasemanifestations resulting from the activation of JNK pathway by theseCaspases.

The finding of a pro-apoptotic role of TRAF2 and TRAF5 provides amechanism by which several non-death domain containing members of theTNFR family, such as TNFR2, CD40, CD30 (GenBank P28908) and LTPR, maymediate cell death. These receptors have been shown to bind to TRAFproteins, but the mechanism by which they mediate cell death has notbeen elucidated. The TRAF proteins have been found to directly bind toCaspase 7, 8 and 10, thus providing a molecular framework for themediation of cell death by these receptors. Therefore, agents that canblock the interaction of TRAF proteins with Caspases may be used for thetreatment of diseases resulting from the dysfunction/overactivity ofthese receptors. These agents might include dominant-negative mutants ofCaspases, cell permeable peptide homologs, small molecule compounds orantisense agents.

One of the proteins encoded by the Kaposi sarcoma associated HerpesVirus (K13-ORF) can directly interact with TRAF proteins andserine-threonine protein kinases and activate the NF-κB pathway. Thisfinding is of critical importance for the development of therapeuticagents against the various malignancies caused by this agent. Thisfinding is of special interest because Epstein-Barr Virus (EBV), anotherherpes virus that has been associated with several human malignancies,has been shown to produce a protein which interacts with the TRAFproteins. Agents which interfere with the interaction of K134-ORF withthe TRAF proteins may prove to be effective therapeutic agents againstKS, multiple myeloma and other malignancies associated with KSHV and forwhich no effective therapy is available at present.

MC159L, a DEDs-containing protein encoded by the Molluscum Contagiosumvirus, binds to TRAF2 but does not activate NF-κB. Moreover, MC159L isan effective inhibitor of NF-κB activation mediated by Caspases. As lackof an inflammatory response to the virus plays a crucial role in thepersistence and recurrence of the molluscum contagiosum infection,agents blocking the activity of MC159L may be used to elicit an immuneresponse to this infection. Such agents could include peptides, smallmolecules or DNA fragments encoding antisense or dominant negativeinhibitors. Due to the cutaneous nature of this infection, it will berelatively easy to deliver the above agents to the target lesion using anumber of currently available drug/gene delivery technologies.Alternatively, MC159L or its analogs may be used as anti-inflammatoryagents for the treatment of diseases caused by undesirable inflammatoryresponse such as rheumatoid arthritis, cerebral/myocardial infarctionand septic shock.

It has been found that all dual DEDs-containing proteins interact withTRAF family members. In addition to KSHV and MC159L, suchDEDs-containing proteins have been discovered in other viruses such asherpes virus simirii, bovine herpes virus and equine herpes virus. As aresult of large scale genomic sequencing, such dual DEDs-containingproteins are likely to be discovered in other viruses of clinicalimportance in future.

Evidence is provided that Caspase 7, 8, 10, MRIT-α1 and DEDs-containingviral open reading frames can directly interact with NIK, RIP, IKK1 andIKK2. Moreover, IKK1 can interact with RIP. Thus, a TRAF-independentpathway has been identified for the activation of NF-κB. Knowledge ofthis pathway can be used to develop highly specific inhibitors of NF-κBactivation mediated by various members of the TNFR family.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 40                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 78                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - Met Ser Ala Glu Val Ile His Gln Val Glu Gl - #u Ala Leu Asp Thr        Asp                                                                               1               5 - #                 10 - #                 15             - - Glu Lys Glu Met Leu Leu Phe Leu Cys Arg As - #p Val Ala Ile Asp Val                   20     - #             25     - #             30                  - - Val Pro Pro Asn Val Arg Asp Leu Leu Asp Il - #e Leu Arg Glu Arg Gly               35         - #         40         - #         45                      - - Lys Leu Ser Val Gly Asp Leu Ala Glu Leu Le - #u Tyr Arg Val Arg Arg           50             - #     55             - #     60                          - - Phe Asp Leu Leu Lys Arg Ile Leu Lys Met As - #p Arg Lys Ala               65                 - # 70                 - # 75                              - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 84                                                              <212> TYPE: PRT                                                               <213> ORGANISM: HUMAN HERPESVIRUS 8                                            - - <400> SEQUENCE: 2                                                         - - Val Ser Asp Tyr Arg Val Leu Met Ala Glu Il - #e Gly Glu Asp Leu Asp        1               5 - #                 10 - #                 15              - - Lys Ser Asp Val Ser Ser Leu Ile Phe Leu Me - #t Lys Asp Tyr Met Gly                   20     - #             25     - #             30                  - - Arg Gly Lys Ile Ser Leu Glu Leu Ser Phe Le - #u Asp Leu Val Val Glu               35         - #         40         - #         45                      - - Leu Glu Lys Leu Asn Leu Val Ala Pro Asp Gl - #n Leu Asp Leu Leu Glu           50             - #     55             - #     60                          - - Lys Cys Leu Lys Asn Ile His Arg Ile Asp Le - #u Lys Thr Lys Ile Gln       65                 - # 70                 - # 75                 - # 80       - - Lys Tyr Lys Gln                                                           - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 79                                                              <212> TYPE: PRT                                                               <213> ORGANISM: HUMAN HERPESVIRUS 8                                            - - <400> SEQUENCE: 3                                                         - - Met Ala Thr Tyr Glu Val Leu Cys Glu Val Al - #a Arg Lys Leu Gly Thr        1               5 - #                 10 - #                 15              - - Asp Asp Arg Glu Val Val Leu Phe Leu Leu As - #n Val Phe Ile Pro Gln                   20     - #             25     - #             30                  - - Pro Thr Leu Ala Gln Leu Ile Gly Ala Leu Ar - #g Ala Leu Lys Glu Glu               35         - #         40         - #         45                      - - Gly Arg Leu Thr Phe Pro Leu Leu Ala Glu Cy - #s Leu Phe Arg Ala Gly           50             - #     55             - #     60                          - - Arg Arg Asp Leu Leu Arg Asp Leu Leu His Le - #u Asp Pro Arg Phe           65                 - # 70                 - # 75                              - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Equine Herpesvirus                                             - - <400> SEQUENCE: 4                                                         - - Phe Ser Pro Tyr Gln Leu Thr Val Leu His Va - #l Asp Gly Glu Leu Cys        1               5 - #                 10 - #                 15              - - Ala Arg Asp Ile Arg Ser Leu Ile Phe Leu Se - #r Lys Asp Thr Ile Gly                   20     - #             25     - #             30                  - - Ser Arg Ser Thr Pro Gln Thr Ser Tyr Thr Gl - #y Cys Thr Val Trp Leu               35         - #         40         - #         45                      - - Thr                                                                       - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 79                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Equine Herpesvirus                                             - - <400> SEQUENCE: 5                                                         - - Met Ser His Tyr Ser Met Ile Asp Thr Tyr Ph - #e Ser Leu Asp Glu Asp        1               5 - #                 10 - #                 15              - - Glu Thr Glu Thr Tyr Leu Tyr Leu Cys Arg As - #p Leu Leu Lys Asn Lys                   20     - #             25     - #             30                  - - Gly Glu Phe Gln Cys Thr Arg Asp Ala Phe Ly - #s Phe Leu Ser Asp Tyr               35         - #         40         - #         45                      - - Ala Cys Leu Ser Ala Ala Asn Gln Met Glu Le - #u Leu Phe Ala Val Gly           50             - #     55             - #     60                          - - Arg Leu Asp Leu Ile Arg Arg Ile Phe Gly Gl - #n Thr Trp Thr Pro           65                 - # 70                 - # 75                              - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 82                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 6                                                         - - Cys Ser Pro Phe Arg Cys Leu Met Ala Leu Va - #l Asn Asp Phe Leu Ser        1               5 - #                 10 - #                 15              - - Asp Leu Glu Val Glu Glu Met Tyr Phe Leu Cy - #s Ala Pro Arg Leu Glu                   20     - #             25     - #             30                  - - Ser His Leu Glu Pro Gly Ser Lys Lys Ser Ph - #e Leu Arg Leu Ala Ser               35         - #         40         - #         45                      - - Leu Leu Glu Asp Leu Glu Leu Leu Gly Gly As - #p Lys Leu Thr Phe Leu           50             - #     55             - #     60                          - - Arg His Leu Leu Thr Thr Ile Gly Arg Ala As - #p Leu Val Lys Asn Leu       65                 - # 70                 - # 75                 - # 80       - - Gln Val                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 82                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 7                                                         - - Met Ser Asp Ser Lys Glu Val Pro Ser Leu Pr - #o Phe Leu Arg His Leu        1               5 - #                 10 - #                 15              - - Leu Glu Glu Leu Asp Ser His Glu Asp Ser Le - #u Leu Leu Phe Leu Cys                   20     - #             25     - #             30                  - - His Asp Ala Ala Pro Gly Cys Thr Thr Val Th - #r Gln Ala Leu Cys Ser               35         - #         40         - #         45                      - - Leu Ser Gln Gln Arg Lys Leu Thr Leu Ala Al - #a Leu Val Glu Met Leu           50             - #     55             - #     60                          - - Tyr Val Leu Gln Arg Met Asp Leu Leu Lys Se - #r Arg Phe Gly Leu Ser       65                 - # 70                 - # 75                 - # 80       - - Lys Glu                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 86                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 8                                                         - - Leu Thr Arg Tyr Arg Lys Leu Met Val Cys Va - #l Gly Glu Glu Leu Asp        1               5 - #                 10 - #                 15              - - Ser Ser Glu Leu Arg Ala Leu Arg Leu Phe Al - #a Cys Asn Leu Asn Pro                   20     - #             25     - #             30                  - - Ser Leu Ser Thr Ala Leu Ser Glu Ser Ser Ar - #g Phe Val Glu Leu Val               35         - #         40         - #         45                      - - Leu Ala Leu Glu Asn Val Gly Leu Val Ser Pr - #o Ser Ser Val Ser Val           50             - #     55             - #     60                          - - Leu Ala Asp Met Leu Arg Thr Leu Arg Arg Le - #u Asp Leu Cys Gln Gln       65                 - # 70                 - # 75                 - # 80       - - Leu Val Glu Tyr Glu Gln                                                                   85                                                            - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 79                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 9                                                         - - Met Ala His Glu Pro Ile Pro Phe Ser Phe Le - #u Arg Asn Leu Leu Ala        1               5 - #                 10 - #                 15              - - Glu Leu Asp Ala Ser Glu His Glu Val Leu Ar - #g Phe Leu Cys Arg Asp                   20     - #             25     - #             30                  - - Val Ala Pro Ala Ser Lys Thr Ala Glu Asp Al - #a Leu Arg Ala Leu Gln               35         - #         40         - #         45                      - - Arg Arg Arg Leu Leu Thr Leu Ser Ser Met Al - #a Glu Leu Leu Cys Ala           50             - #     55             - #     60                          - - Leu Arg Arg Phe Asp Val Leu Lys Val Arg Ph - #e Gly Met Thr Arg           65                 - # 70                 - # 75                              - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 82                                                              <212> TYPE: PRT                                                               <213> ORGANISM: 'Axial Seamount' polynoid polyc - #haete                       - - <400> SEQUENCE: 10                                                        - - Leu Ser Gln Tyr Arg Leu Gln Val Ala Ala Il - #e Asn Asn Met Val Gly        1               5 - #                 10 - #                 15              - - Ser Glu Asp Leu Arg Val Met Cys Leu Cys Al - #a Gly Lys Leu Leu Pro                   20     - #             25     - #             30                  - - Pro Ser Cys Thr Pro Arg Cys Leu Val Asp Le - #u Val Ser Ala Leu Glu               35         - #         40         - #         45                      - - Asp Ala Gly Ala Ile Ser Pro Gln Asp Val Se - #r Val Leu Val Thr Leu           50             - #     55             - #     60                          - - Leu His Ala Val Cys Arg Tyr Asp Leu Ser Va - #l Ala Leu Ser Ala Val       65                 - # 70                 - # 75                 - # 80       - - Ala His                                                                   - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 83                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl - #y Glu Lys Leu Asp Ser        1               5 - #                 10 - #                 15              - - Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le - #u Asp Tyr Ile Pro Gln                   20     - #             25     - #             30                  - - Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me - #t Leu Phe Gln Arg Leu               35         - #         40         - #         45                      - - Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le - #u Ser Phe Leu Lys Glu           50             - #     55             - #     60                          - - Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Le - #u Ile Thr Tyr Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Thr Arg Lys                                                               - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 83                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 12                                                        - - Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Il - #e Ser Glu Glu Val Ser        1               5 - #                 10 - #                 15              - - Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Le - #u Gln Glu Glu Ile Ser                   20     - #             25     - #             30                  - - Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Le - #u Asp Ile Phe Ile Glu               35         - #         40         - #         45                      - - Met Glu Lys Arg Val Ile Leu Gly Glu Gly Ly - #s Leu Asp Ile Leu Lys           50             - #     55             - #     60                          - - Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Le - #u Lys Ile Ile Asn Asp       65                 - # 70                 - # 75                 - # 80       - - Tyr Glu Glu                                                               - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 95                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 13                                                        - - Met Lys Ser Gln Gly Gln His Trp Tyr Ser Se - #r Ser Asp Lys Asn Cys        1               5 - #                 10 - #                 15              - - Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Il - #e Asp Ser Asn Leu Gly                   20     - #             25     - #             30                  - - Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cy - #s Ile Gly Leu Val Pro               35         - #         40         - #         45                      - - Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Se - #r Asp Val Phe Glu His           50             - #     55             - #     60                          - - Leu Leu Ala Gly Asp Leu Leu Ser Glu Glu As - #p Pro Phe Phe Leu Ala       65                 - # 70                 - # 75                 - # 80       - - Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Le - #u Leu Gln His Leu                           85 - #                 90 - #                 95              - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 80                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 14                                                        - - Val Ser Leu Phe Arg Asn Leu Leu Tyr Glu Le - #u Ser Glu Gly Ile Asp        1               5 - #                 10 - #                 15              - - Ser Glu Asn Leu Lys Asp Met Ile Phe Leu Le - #u Lys Asp Ser Leu Pro                   20     - #             25     - #             30                  - - Lys Thr Glu Met Thr Ser Leu Ser Phe Leu Al - #a Phe Leu Glu Lys Gln               35         - #         40         - #         45                      - - Gly Lys Ile Asp Glu Asp Asn Leu Thr Cys Le - #u Glu Asp Leu Cys Lys           50             - #     55             - #     60                          - - Thr Val Val Pro Lys Leu Leu Arg Asn Ile Gl - #u Lys Tyr Lys Arg Glu       65                 - # 70                 - # 75                 - # 80       - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 83                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 15                                                        - - Met Asp Pro Phe Leu Val Leu Leu His Ser Va - #l Ser Ser Ser Leu Ser        1               5 - #                 10 - #                 15              - - Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cy - #s Leu Gly Arg Val Gly                   20     - #             25     - #             30                  - - Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Le - #u Asp Leu Phe Ser Met               35         - #         40         - #         45                      - - Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly Hi - #s Thr Glu Leu Leu Arg           50             - #     55             - #     60                          - - Glu Leu Leu Ala Ser Leu Arg Arg His Asp Le - #u Leu Arg Arg Val Asp       65                 - # 70                 - # 75                 - # 80       - - Asp Phe Glu                                                               - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 83                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 16                                                        - - Met Val Glu Tyr Gly Thr Leu Phe Gln Asp Le - #u Thr Asn Asn Ile Thr        1               5 - #                 10 - #                 15              - - Leu Glu Asp Leu Glu Gln Leu Lys Ser Ala Cy - #s Lys Glu Asp Ile Pro                   20     - #             25     - #             30                  - - Ser Glu Lys Ser Glu Glu Ile Thr Thr Gly Se - #r Ala Trp Phe Ser Phe               35         - #         40         - #         45                      - - Leu Glu Ser His Asn Lys Leu Asp Lys Asp As - #n Leu Ser Ile Ile Glu           50             - #     55             - #     60                          - - His Ile Phe Glu Ile Ser Arg Arg Pro Asp Le - #u Leu Thr Met Val Val       65                 - # 70                 - # 75                 - # 80       - - Asp Tyr Arg                                                               - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 221                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 17                                                        - - Met Ser Ala Glu Val Ile His Gln Val Glu Gl - #u Ala Leu Asp Thr Asp        1               5 - #                 10 - #                 15              - - Glu Lys Glu Met Leu Leu Phe Leu Cys Arg As - #p Val Ala Ile Asp Val                   20     - #             25     - #             30                  - - Val Pro Pro Asn Val Arg Asp Leu Leu Asp Il - #e Leu Arg Glu Arg Gly               35         - #         40         - #         45                      - - Lys Leu Ser Val Gly Asp Leu Ala Glu Leu Le - #u Tyr Arg Val Arg Arg           50             - #     55             - #     60                          - - Phe Asp Leu Leu Lys Arg Ile Leu Lys Met As - #p Arg Lys Ala Val Glu       65                 - # 70                 - # 75                 - # 80       - - Thr His Leu Leu Arg Asn Pro His Leu Val Se - #r Asp Tyr Arg Val Leu                       85 - #                 90 - #                 95              - - Met Ala Glu Ile Gly Glu Asp Leu Asp Lys Se - #r Asp Val Ser Ser Leu                  100      - #           105      - #           110                  - - Ile Phe Leu Met Lys Asp Tyr Met Gly Arg Gl - #y Lys Ile Ser Lys Glu              115          - #       120          - #       125                      - - Lys Ser Phe Leu Asp Leu Val Val Glu Leu Gl - #u Lys Leu Asn Leu Val          130              - #   135              - #   140                          - - Ala Pro Asp Gln Leu Asp Leu Leu Glu Lys Cy - #s Leu Lys Asn Ile His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Ile Asp Leu Lys Thr Lys Ile Gln Lys Ty - #r Lys Gln Ser Val        Gln                                                                                             165  - #               170  - #               175             - - Gly Ala Gly Thr Ser Tyr Arg Asn Val Leu Gl - #n Ala Ala Ile Gln Lys                  180      - #           185      - #           190                  - - Ser Leu Lys Asp Pro Ser Asn Asn Phe Arg Me - #t Ile Thr Pro Tyr Ala              195          - #       200          - #       205                      - - His Cys Pro Asp Leu Lys Ile Leu Gly Asn Cy - #s Ser Met                      210              - #   215              - #   220                          - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 180                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 18                                                        - - Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl - #y Glu Gln Leu Asp Ser        1               5 - #                 10 - #                 15              - - Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le - #u Asp Tyr Ile Pro Gln                   20     - #             25     - #             30                  - - Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me - #t Leu Phe Gln Arg Leu               35         - #         40         - #         45                      - - Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le - #u Ser Phe Leu Lys Glu           50             - #     55             - #     60                          - - Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Le - #u Ile Thr Tyr Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl - #n Thr Pro Gly Arg Ala                       85 - #                 90 - #                 95             Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gl - #n Ile Ser Glu Glu Val                       100      - #           105      - #           110                  - - Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le - #u Leu Gln Glu Glu Ile              115          - #       120          - #       125                      - - Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le - #u Leu Asp Ile Phe Ile          130              - #   135              - #   140                          - - Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gl - #y Lys Leu Asp Ile Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le - #u Leu Lys Ile Ile        Asn                                                                                             165  - #               170  - #               175             - - Asp Tyr Glu Glu                                                                      180                                                                - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 208                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 19                                                        - - Met Asp Pro Phe Leu Val Leu Leu His Ser Va - #l Ser Ser Ser Leu Ser        1               5 - #                 10 - #                 15              - - Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cy - #s Leu Gly Arg Val Gly                   20     - #             25     - #             30                  - - Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Le - #u Asp Leu Phe Ser Met               35         - #         40         - #         45                      - - Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly Hi - #s Thr Glu Leu Leu Arg           50             - #     55             - #     60                          - - Glu Leu Leu Ala Ser Leu Arg Arg His Asp Le - #u Leu Arg Arg Val Asp       65                 - # 70                 - # 75                 - # 80       - - Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Al - #a Pro Gly Glu Glu Asp                       85 - #                 90 - #                 95             Leu Cys Ala Ala Phe Asn Val Ile Cys Asp As - #n Asp Gly Lys Asp Trp                       100      - #           105      - #           110                  - - Arg Arg Leu Ala Arg Gln Leu Lys Val Ser As - #p Thr Lys Ile Asp Ser              115          - #       120          - #       125                      - - Ile Glu Asp Arg Tyr Pro Arg Asn Leu Thr Gl - #u Arg Val Arg Glu Ser          130              - #   135              - #   140                          - - Leu Arg Ile Trp Lys Asn Thr Glu Lys Glu As - #n Ala Thr Val Ala His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Val Gly Ala Leu Arg Ser Cys Gln Met As - #n Leu Val Ala Asp        Leu                                                                                             165  - #               170  - #               175             - - Val Gln Glu Val Gln Gln Ala Arg Asp Leu Gl - #n Asn Arg Ser Gly Ala                  180      - #           185      - #           190                  - - Met Ser Pro Met Ser Trp Asn Ser Asp Ala Se - #r Thr Ser Glu Ala Ser              195          - #       200          - #       205                      - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 139                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 20                                                        - - Met Ala Thr Tyr Glu Val Leu Cys Glu Val Al - #a Arg Lys Leu Gly Thr        1               5 - #                 10 - #                 15              - - Asp Asp Arg Glu Val Val Leu Phe Leu Leu As - #n Val Phe Ile Pro Gln                   20     - #             25     - #             30                  - - Pro Thr Leu Ala Gln Leu Ile Gly Ala Leu Ar - #g Ala Leu Lys Glu Glu               35         - #         40         - #         45                      - - Gly Arg Leu Thr Phe Pro Leu Leu Ala Glu Cy - #s Leu Phe Arg Ala Gly           50             - #     55             - #     60                          - - Arg Arg Asp Leu Leu Arg Asp Leu Leu His Le - #u Asp Pro Arg Phe Leu       65                 - # 70                 - # 75                 - # 80       - - Glu Arg His Leu Ala Gly Thr Met Ser Tyr Ph - #e Ser Pro Tyr Gln Leu                       85 - #                 90 - #                 95              - - Thr Val Leu His Val Asp Gly Glu Leu Cys Al - #a Arg Asp Ile Arg Ser                  100      - #           105      - #           110                  - - Leu Ile Phe Leu Ser Lys Asp Thr Ile Gly Se - #r Arg Ser Thr Pro Gln              115          - #       120          - #       125                      - - Thr Ser Tyr Thr Gly Cys Thr Val Trp Lys Th - #r                              130              - #   135                                                 - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 241                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 21                                                        - - Met Ser Asp Ser Lys Glu Val Pro Ser Leu Pr - #o Phe Leu Arg His Leu        1               5 - #                 10 - #                 15              - - Leu Glu Glu Leu Asp Ser His Glu Asp Ser Le - #u Leu Leu Phe Leu Cys                   20     - #             25     - #             30                  - - His Asp Ala Ala Pro Gly Cys Thr Thr Val Th - #r Gln Ala Leu Cys Ser               35         - #         40         - #         45                      - - Leu Ser Gln Gln Arg Lys Leu Thr Leu Ala Al - #a Leu Val Glu Met Leu           50             - #     55             - #     60                          - - Tyr Val Leu Gln Arg Met Asp Leu Leu Lys Se - #r Arg Phe Gly Leu Ser       65                 - # 70                 - # 75                 - # 80       - - Lys Glu Gly Ala Glu Gln Leu Leu Gly Thr Se - #r Phe Leu Thr Arg Tyr                       85 - #                 90 - #                 95              - - Arg Lys Leu Met Val Cys Val Gly Glu Glu Le - #u Asp Ser Ser Glu Leu                  100      - #           105      - #           110                  - - Arg Ala Leu Arg Leu Phe Ala Cys Asn Leu As - #n Pro Ser Leu Ser Thr              115          - #       120          - #       125                      - - Ala Leu Ser Glu Ser Ser Arg Phe Val Glu Le - #u Val Leu Ala Leu Glu          130              - #   135              - #   140                          - - Asn Val Gly Leu Val Ser Pro Ser Ser Val Se - #r Val Leu Ala Asp Met      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Arg Thr Leu Arg Arg Leu Asp Leu Cys Gl - #n Gln Leu Val Glu        Tyr                                                                                             165  - #               170  - #               175             - - Glu Gln Gln Glu Gln Ala Arg Tyr Arg Tyr Cy - #s Tyr Ala Ala Ser Pro                  180      - #           185      - #           190                  - - Ser Leu Pro Val Arg Thr Leu Arg Arg Gly Hi - #s Gly Ala Ser Glu His              195          - #       200          - #       205                      - - Glu Gln Leu Cys Met Pro Val Gln Glu Ser Se - #r Asp Ser Pro Glu Leu          210              - #   215              - #   220                          - - Leu Arg Thr Pro Val Gln Glu Ser Ser Ser As - #p Ser Pro Glu Gln Thr      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr                                                                       - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 371                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Molluscum contagiosum virus                                    - - <400> SEQUENCE: 22                                                        - - Met Ala His Glu Pro Ile Pro Phe Ser Phe Le - #u Arg Asn Leu Leu        Ala                                                                               1               5 - #                 10 - #                 15             - - Glu Leu Asp Ala Ser Glu His Glu Val Leu Ar - #g Phe Leu Cys Arg Asp                   20     - #             25     - #             30                  - - Val Ala Pro Ala Ser Lys Thr Ala Glu Asp Al - #a Leu Arg Ala Leu Gln               35         - #         40         - #         45                      - - Arg Arg Arg Leu Leu Thr Leu Ser Ser Met Al - #a Glu Leu Leu Cys Ala           50             - #     55             - #     60                          - - Leu Arg Arg Phe Asp Val Leu Lys Val Arg Ph - #e Gly Met Thr Arg Glu       65                 - # 70                 - # 75                 - # 80       - - Cys Ala Gly Arg Leu Leu Gly His Gly Phe Le - #u Ser Gln Tyr Arg Leu                       85 - #                 90 - #                 95              - - Gln Val Ala Ala Ile Asn Asn Met Val Gly Se - #r Glu Asp Leu Arg Val                  100      - #           105      - #           110                  - - Met Cys Leu Cys Ala Gly Lys Leu Leu Pro Pr - #o Ser Cys Thr Pro Arg              115          - #       120          - #       125                      - - Cys Leu Val Asp Leu Val Ser Ala Leu Glu As - #p Ala Gly Ala Ile Ser          130              - #   135              - #   140                          - - Pro Gln Asp Val Ser Val Leu Val Thr Leu Le - #u His Ala Val Cys Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Asp Leu Ser Val Ala Leu Ser Ala Val Al - #a His Gly His Met        Thr                                                                                             165  - #               170  - #               175             - - Val Gly Val Gly Thr Pro Val Gln Asp Glu Pr - #o Met Asp Val Leu Glu                  180      - #           185      - #           190                  - - Val Asp Asp Ala Glu Pro Met Glu Ala Thr Pr - #o Ala Cys Asp Glu Ile              195          - #       200          - #       205                      - - Gly Val Val Lys Leu Ala Gly Ala Ala Ser Al - #a Gly Ala Pro Leu Ala          210              - #   215              - #   220                          - - Asp Gly Ala Phe Ala Ala Cys Thr Ser Ala Gl - #y Lys Gly Glu Asp Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Thr Ser Asp Leu Thr Asp Ser Glu Pro Gl - #u Asp Ser Val Phe        Ala                                                                                             245  - #               250  - #               255             - - Val Ala Asp Pro Val Tyr Ala Asp Val Asp Le - #u Ser Met Phe Val Arg                  260      - #           265      - #           270                  - - Ala Asn Ala Thr Ala Asp Ser Ser Met Phe Va - #l Asn Ala Asp Ala Gly              275          - #       280          - #       285                      - - Ala Asp Ser Ser Leu Val Asn Ala Asp Ala Gl - #y Ala Asp Ser Ser Leu          290              - #   295              - #   300                          - - Val Asn Ala Asp Ala Gly Ala Asp Ser Ser Le - #u Val Asn Ala Val Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Ala Asn Ser Ser Leu Met Arg Thr Thr Se - #r Ala Cys Thr Asp        Ser                                                                                             325  - #               330  - #               335             - - Glu Pro Glu Asp Ser Ala Gly Pro Ser Cys Al - #a Gly Met Ala Leu Ser                  340      - #           345      - #           350                  - - Met Phe Gly Arg Ala Lys Ser Val Ser Ser Le - #u Leu Leu Arg Thr Lys              355          - #       360          - #       365                      - - Ala Ser Thr                                                                  370                                                                        - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 171                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Equine Herpesvirus                                             - - <400> SEQUENCE: 23                                                        - - Met Ser His Tyr Ser Met Ile Asp Thr Tyr Ph - #e Ser Leu Asp Glu Asp        1               5 - #                 10 - #                 15              - - Glu Thr Glu Thr Tyr Leu Tyr Leu Cys Arg As - #p Leu Leu Lys Asn Lys                   20     - #             25     - #             30                  - - Gly Glu Phe Gln Cys Thr Arg Asp Ala Phe Ly - #s Phe Leu Ser Asp Tyr               35         - #         40         - #         45                      - - Ala Cys Leu Ser Ala Ala Asn Gln Met Glu Le - #u Leu Phe Arg Val Gly           50             - #     55             - #     60                          - - Arg Leu Asp Leu Ile Arg Arg Ile Phe Gly Gl - #n Thr Trp Thr Pro Asp       65                 - # 70                 - # 75                 - # 80       - - Ser Cys Pro Arg Tyr Tyr Met Pro Ile Cys Se - #r Pro Phe Arg Cys Leu                       85 - #                 90 - #                 95              - - Met Ala Leu Val Asn Asp Phe Leu Ser Asp Le - #u Glu Val Glu Glu Met                  100      - #           105      - #           110                  - - Tyr Phe Leu Cys Ala Pro Arg Leu Glu Ser Hi - #s Leu Glu Pro Gly Ser              115          - #       120          - #       125                      - - Lys Lys Ser Phe Leu Arg Leu Ala Ser Leu Le - #u Glu Asp Leu Glu Leu          130              - #   135              - #   140                          - - Leu Gly Gly Asp Lys Leu Thr Phe Leu Arg Hi - #s Leu Leu Thr Thr Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Arg Ala Asp Leu Val Lys Asn Leu Gln Va - #l                                          165  - #               170                                     - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 130                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 24                                                        - - Met Val Glu Tyr Gly Thr Leu Phe Gln Asp Le - #u Thr Asn Asn Ile        Thr                                                                               1               5 - #                 10 - #                 15             - - Leu Glu Asp Leu Glu Gln Leu Lys Ser Ala Cy - #s Lys Glu Asp Ile Pro                   20     - #             25     - #             30                  - - Ser Glu Lys Ser Glu Glu Ile Thr Thr Gly Se - #r Ala Trp Phe Ser Phe               35         - #         40         - #         45                      - - Leu Glu Ser His Asn Lys Leu Asp Lys Asp As - #n Leu Ser Ile Ile Glu           50             - #     55             - #     60                          - - His Ile Phe Glu Ile Ser Arg Arg Pro Asp Le - #u Leu Thr Met Val Val       65                 - # 70                 - # 75                 - # 80       - - Asp Tyr Arg Thr Arg Val Leu Lys Ile Ser Gl - #u Glu Asp Glu Leu Asp                       85 - #                 90 - #                 95              - - Thr Lys Leu Thr Arg Ile Pro Ser Ala Lys Ly - #s Tyr Lys Asp Ile Ile                  100      - #           105      - #           110                  - - Arg Gln Pro Ser Glu Glu Glu Ile Ile Lys Le - #u Gly Pro Pro Pro Lys              115          - #       120          - #       125                      - - Lys Ala                                                                      130                                                                        - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 210                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 25                                                        - - Met Lys Ser Gln Gly Gln His Trp Tyr Ser Se - #r Ser Asp Lys Asn Cys        1               5 - #                 10 - #                 15              - - Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Il - #e Asp Ser Asn Leu Gly                   20     - #             25     - #             30                  - - Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cy - #s Ile Gly Leu Val Pro               35         - #         40         - #         45                      - - Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Se - #r Asp Val Phe Glu His           50             - #     55             - #     60                          - - Leu Leu Ala Gly Asp Leu Leu Ser Glu Glu As - #p Pro Phe Phe Leu Ala       65                 - # 70                 - # 75                 - # 80       - - Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Le - #u Leu Gln His Leu Asn                       85 - #                 90 - #                 95              - - Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pr - #o Thr Arg Gln Arg Val                  100      - #           105      - #           110                  - - Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Se - #r Glu Gly Ile Asp Ser              115          - #       120          - #       125                      - - Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Ly - #s Asp Ser Leu Pro Lys          130              - #   135              - #   140                          - - Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Ph - #e Leu Glu Lys Gln Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Gl - #u Asp Leu Cys Lys        Thr                                                                                             165  - #               170  - #               175             - - Val Val Pro Lys Leu Leu Arg Asn Ile Glu Ly - #s Tyr Lys Arg Glu Lys                  180      - #           185      - #           190                  - - Ala Ile Gln Ile Val Thr Pro Pro Val Asp Ly - #s Glu Ala Glu Ser Tyr              195          - #       200          - #       205                      - - Gln Gly                                                                      210                                                                        - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 479                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 26                                                        - - Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl - #y Glu Gln Leu Asp Ser        1               5 - #                 10 - #                 15              - - Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le - #u Asp Tyr Ile Pro Gln                   20     - #             25     - #             30                  - - Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me - #t Leu Phe Gln Arg Leu               35         - #         40         - #         45                      - - Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le - #u Ser Phe Leu Lys Glu           50             - #     55             - #     60                          - - Leu Leu Phe Arg Ile Asn Arg Leu Ala Leu Le - #u Ile Thr Tyr Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl - #n Thr Pro Gly Arg Ala                       85 - #                 90 - #                 95              - - Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gl - #n Ile Ser Glu Glu Val                  100      - #           105      - #           110                  - - Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le - #u Leu Gln Glu Glu Ile              115          - #       120          - #       125                      - - Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le - #u Leu Asp Ile Phe Ile          130              - #   135              - #   140                          - - Glu Met Ile Lys Arg Val Ile Leu Gly Glu Gl - #y Lys Leu Asp Ile Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le - #u Leu Lys Ile Ile        Asn                                                                                             165  - #               170  - #               175             - - Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Se - #r Ser Leu Glu Gly Ser                  180      - #           185      - #           190                  - - Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cy - #s Gly Val Met Thr Ile              195          - #       200          - #       205                      - - Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Se - #r Gln Thr Leu Asp Lys          210              - #   215              - #   220                          - - Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Ty - #r Cys Leu Ile Ile Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn His Asn Phe Ala Lys Ala Arg Glu Lys Va - #l Pro Lys Leu His        Ser                                                                                             245  - #               250  - #               255             - - Ile Arg Asp Arg Asn Gly Thr His Leu Asp Al - #a Gly Ala Leu Thr Thr                  260      - #           265      - #           270                  - - Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pr - #o His Asp Asp Cys Thr              275          - #       280          - #       285                      - - Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Ty - #r Gln Leu Met Asp His          290              - #   295              - #   300                          - - Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Le - #u Ser His Gly Asp Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Pr - #o Pro Ile Tyr Glu        Leu                                                                                             325  - #               330  - #               335             - - Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Se - #r Leu Ala Gly Lys Pro                  340      - #           345      - #           350                  - - Lys Val Phe Phe Ile Gln Ala Cys Gln Gly As - #p Asn Tyr Gln Lys Gly              355          - #       360          - #       365                      - - Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pr - #o Tyr Leu Glu Met Asp          370              - #   375              - #   380                          - - Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro As - #p Glu Ala Asp Phe Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Gly Met Ala Thr Val Asn Asn Cys Val Se - #r Tyr Arg Asn Pro        Ala                                                                                             405  - #               410  - #               415             - - Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gl - #n Ser Leu Arg Glu Arg                  420      - #           425      - #           430                  - - Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Le - #u Thr Glu Val Asn Tyr              435          - #       440          - #       445                      - - Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Me - #t Gly Lys Gln Met Pro          450              - #   455              - #   460                          - - Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Va - #l Phe Pro Ser Asp          465                 4 - #70                 4 - #75                            - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 479                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 27                                                        - - Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl - #y Glu Gln Leu Asp Ser        1               5 - #                 10 - #                 15              - - Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le - #u Asp Tyr Ile Pro Gln                   20     - #             25     - #             30                  - - Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me - #t Leu Phe Gln Arg Leu               35         - #         40         - #         45                      - - Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le - #u Ser Phe Leu Lys Glu           50             - #     55             - #     60                          - - Leu Leu Phe Arg Ile Asn Arg Leu Asp Ala Le - #u Ile Thr Tyr Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl - #n Thr Pro Gly Arg Ala                       85 - #                 90 - #                 95              - - Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gl - #n Ile Ser Glu Glu Val                  100      - #           105      - #           110                  - - Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le - #u Leu Gln Glu Glu Ile              115          - #       120          - #       125                      - - Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le - #u Leu Asp Ile Phe Ile          130              - #   135              - #   140                          - - Glu Asn Glu Lys Arg Val Ile Leu Gly Glu Gl - #y Lys Leu Asp Ile Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le - #u Leu Lys Ile Ile        Asn                                                                                             165  - #               170  - #               175             - - Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Se - #r Ser Leu Glu Gly Ser                  180      - #           185      - #           190                  - - Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cy - #s Gly Val Met Thr Ile              195          - #       200          - #       205                      - - Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Se - #r Gln Thr Leu Asp Lys          210              - #   215              - #   220                          - - Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Ty - #r Cys Leu Ile Ile Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn His Asn Phe Ala Lys Ala Arg Glu Lys Va - #l Pro Lys Leu His        Ser                                                                                             245  - #               250  - #               255             - - Ile Arg Asp Arg Asn Gly Thr His Leu Asp Al - #a Gly Ala Leu Thr Thr                  260      - #           265      - #           270                  - - Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pr - #o His Asp Asp Cys Thr              275          - #       280          - #       285                      - - Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Ty - #r Gln Leu Met Asp His          290              - #   295              - #   300                          - - Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Le - #u Ser His Gly Asp Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Pr - #o Pro Ile Tyr Glu        Leu                                                                                             325  - #               330  - #               335             - - Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Se - #r Leu Ala Gly Lys Pro                  340      - #           345      - #           350                  - - Lys Val Phe Phe Ile Gln Ala Cys Gln Gly As - #p Asn Tyr Gln Lys Gly              355          - #       360          - #       365                      - - Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pr - #o Tyr Leu Glu Met Asp          370              - #   375              - #   380                          - - Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro As - #p Glu Ala Asp Phe Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Gly Met Ala Thr Val Asn Asn Cys Val Se - #r Tyr Arg Asn Pro        Ala                                                                                             405  - #               410  - #               415             - - Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gl - #n Ser Leu Arg Glu Arg                  420      - #           425      - #           430                  - - Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Le - #u Thr Glu Val Asn Tyr              435          - #       440          - #       445                      - - Glu Asp Ser Asn Lys Asp Asp Lys Lys Asn Me - #t Gly Lys Gln Met Pro          450              - #   455              - #   460                          - - Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Va - #l Phe Pro Ser Asp          465                 4 - #70                 4 - #75                            - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 479                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 28                                                        - - Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl - #y Glu Gln Leu Asp Ser        1               5 - #                 10 - #                 15              - - Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le - #u Asp Tyr Ile Pro Gln                   20     - #             25     - #             30                  - - Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me - #t Leu Phe Gln Arg Leu               35         - #         40         - #         45                      - - Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le - #u Ser Phe Leu Lys Glu           50             - #     55             - #     60                          - - Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Al - #a Ile Thr Tyr Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl - #n Thr Pro Gly Arg Ala                       85 - #                 90 - #                 95              - - Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gl - #n Ile Ser Glu Glu Val                  100      - #           105      - #           110                  - - Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le - #u Leu Gln Glu Glu Ile              115          - #       120          - #       125                      - - Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le - #u Leu Asp Ile Phe Ile          130              - #   135              - #   140                          - - Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gl - #y Lys Leu Asp Ile Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le - #u Leu Lys Ile Ile        Asn                                                                                             165  - #               170  - #               175             - - Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Se - #r Ser Leu Glu Gly Ser                  180      - #           185      - #           190                  - - Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cy - #s Gly Val Met Thr Ile              195          - #       200          - #       205                      - - Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Se - #r Gln Thr Leu Asp Lys          210              - #   215              - #   220                          - - Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Ty - #r Cys Leu Ile Ile Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn His Asn Phe Ala Lys Ala Arg Glu Lys Va - #l Pro Lys Leu His        Ser                                                                                             245  - #               250  - #               255             - - Ile Arg Asp Arg Asn Gly Thr His Leu Asp Al - #a Gly Ala Leu Thr Thr                  260      - #           265      - #           270                  - - Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pr - #o His Asp Asp Cys Thr              275          - #       280          - #       285                      - - Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Ty - #r Gln Leu Met Asp His          290              - #   295              - #   300                          - - Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Le - #u Ser His Gly Asp Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Pr - #o Pro Ile Tyr Glu        Leu                                                                                             325  - #               330  - #               335             - - Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Se - #r Leu Ala Gly Lys Pro                  340      - #           345      - #           350                  - - Lys Val Phe Phe Ile Gln Ala Cys Gln Gly As - #p Asn Tyr Gln Lys Gly              355          - #       360          - #       365                      - - Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pr - #o Tyr Leu Glu Met Asp          370              - #   375              - #   380                          - - Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro As - #p Glu Ala Asp Phe Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Gly Met Ala Thr Val Asn Asn Cys Val Se - #r Tyr Arg Asn Pro        Ala                                                                                             405  - #               410  - #               415             - - Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gl - #n Ser Leu Arg Glu Arg                  420      - #           425      - #           430                  - - Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Le - #u Thr Glu Val Asn Tyr              435          - #       440          - #       445                      - - Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Me - #t Gly Lys Gln Met Pro          450              - #   455              - #   460                          - - Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Va - #l Phe Pro Ser Asp          465                 4 - #70                 4 - #75                            - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 41                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 29                                                        - - gtgtttttta ttcaggctag tcagggggat aactaccaga a    - #                      - #   41                                                                      - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 41                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 30                                                        - - ttctggtagt tatccccctg actagcctga ataaaaaaca c    - #                      - #   41                                                                      - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 31                                                        - - taatagactg gctttgctga ttac          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 32                                                        - - gtaatcagca aagccagtct atta          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 33                                                        - - aatagactgg atgcgctgat tacc          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 34                                                        - - ggtaatcagc gcatccagtc tatt          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 35                                                        - - gactggattt ggcgattacc tacc          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 36                                                        - - ggtaggtaat cgccaaatcc agtc          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 37                                                        - - tcttcattca ggctagccga gggaccgag         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 38                                                        - - ctcggtccct cggctagcct gaatgaaga         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 39                                                        - - ttcatccagg ccagtggtgg ggagc          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 40                                                        - - gctccccacc actggcctgg atgaa          - #                  - #                   25                                                                    __________________________________________________________________________

We claim:
 1. A protein selected from the group consisting of SEQ IDNO:26, SEQ ID NO:27 and SEQ ID NO:28.